ORIGINAL ARTICLE

Resveratrol Protects HUVECs from Oxidized-LDL InducedOxidative Damage by Autophagy Upregulationvia the AMPK/SIRT1 Pathway

Hualei Guo & Yanling Chen & Lizhen Liao & Weikang Wu

Published online: 29 January 2013# Springer Science+Business Media New York 2013

AbstractPurpose Resveratrol could induce basal autophagy throughthe activation of sirtuin. In this study, we investigated theeffect of resveratrol on oxidative injury of human umbilicalendothelial vein cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) and the role of autophagy inthis effect.Methods HUVECs were exposed to 100 mg/L ox-LDLfor 24 h to cause oxidative injury. The effect of differentconcentrations of resveratrol on oxidative damage inHUVECs treated with ox-LDL was evaluated by MTTassay and superoxide dismutase (SOD) activity test. Theautophagic level in different groups was measured by theprotein expression of microtubule-associated protein 1light chain 3 (LC3) and sequestosome 1 (SQSTM1/P62).Autophagosomes were observed under electron micro-scope and fluorescence microscope (by MDC staining).The expression of silencing information regulator1(Sirt1) and AMP activated protein kinaseα1 (AMPK)was investigated by Western blot. Autophagy inhibitor3-methyladenine (3-MA) and Sirt1 inhibitor 6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide (EX527)were used to confirm the role of autophagy in this effectof resveratrol and the pathway involved.Results Resveratrol reversed the decreases in cell viabil-ity (72.9±1.7 % of the control group) and SOD activity(14.37±0.21 U/ml) caused by ox-LDL at 83.4±1.4 % ofthe control group and 16.41±0.27 U/ml respectively.This effect accompanied by upregulation of autophagyand increased protein expression of Sirt1 and AMPKphosphorylation on threonine 172 (p-AMPK). Both 3-

MA and EX527 abolished the protective effect of resver-atrol in cell viability, at 80.4±2.7 % and 73.9±1.1 % ofthe control group respectively. 3-MA inhibited autophagyactivation without any change of Sirt1 expression at boththe mRNA and protein level. EX527 suppressed theexpression of Sirt1 and diminished the upregulation ofautophagy. Addition of 3-MA or EX527 could not affectthe protein level of p-AMPK.Conclusion Resveratrol protected HUVECs from oxida-tive damage caused by ox-LDL. This effect was medi-ated by Sirt1-dependent autophagy via the AMPK/ Sirt1pathway.

Keywords ox-LDL . Oxidative stress . Resveratrol .

Autophagy . AMPK/Sirt1

Introduction

Oxidized low-density lipoprotein (ox-LDL) plays a keyrole in the pathogenesis and plaque rupture of athero-sclerosis through endothelial injury. Endothelial cellsprotect against adhesion of monocytes and platelets andinhibit proliferation of vascular smooth muscle cells [1].Ox-LDL inhibits the expression of constitutive nitricoxide synthase, induces the expression of adhesion mol-ecules and generates reactive oxygen species, leading toendothelial cell apoptosis and foam cell formation [2]. Inendothelial cells, oxidative stress is considered as a ma-jor factor contributing to cell damage. As a result, de-velopment of drugs which protect endothelial cells fromoxidative damage and apoptosis could be a beneficialstrategy for the treatment of atherosclerosis.

Autophagy is a reparative, life-sustaining process bywhich cytoplasmic components are sequestered indouble-membrane vesicles and degraded upon fusion

H. Guo :Y. Chen : L. Liao :W. Wu (*)Department of Pathophysiology, Zhongshan Medical School,Sun Yat-sen University, 74 Zhongshan Er Road,Guangzhou, Guangdong 510080, Chinae-mail: jane__123@163.com

Cardiovasc Drugs Ther (2013) 27:189–198DOI 10.1007/s10557-013-6442-4

with lysosomal compartments [3]. Autophagy is consid-ered to be a house-keeping process by which long-livedproteins and dysfunctional organelles are degraded [4]. Inhuman umbilical endothelial vein cells, autophagy is activatedby ox-LDL, and such activation contributes to the degra-dation of ox-LDL [5]. In atherosclerosis, autophagy isactivated as part of the oxidative stress response bydegrading the damaged material, but in contrast to basalautophagy, excessively stimulated autophagy may causeautophagic death [4, 6].

Resveratrol (RESV, trans-3,5,4′-trihydroxystilbene) isa natural phytoalexin found in grapes and red wine. Agrowing body of evidence indicates that resveratrol hasanti-inflammatory, anti-oxidative, anti-thrombotic andanti-proliferative properties which could explain the car-dioprotective effects observed by a moderate consump-tion of red wine [7]. Resveratrol could improveendothelial function which plays a pivotal role in ath-erosclerosis development [8, 9]. In addition, resveratrolinduces basal autophagy through the inhibition ofmTOR as a sirtuin activator [10, 11]. However, theeffect of resveratrol on the oxidative damage and auto-phagic level in HUVECs exposed to ox-LDL is stillunknown. In the present study, we attempted to explorethis effect and the role of autophagy in it. We alsodelineated the underlying mechanisms with an emphasison the AMPK/Sirt1 pathway.

Materials and Methods

Cell Culture

Primary human umbilical vein endothelial cells(HUVECs) were isolated from human umbilical veinsafter 0.25 % trypsin (Hyclone, USA) digestion and main-tained on gelatin (Acumedia, Lansing, USA) coated T25flasks in serum-free medium (SFM; Gibco, Grand Island,NY) supplemented with 20 % fetal bovine serum (FBS;Hyclone), 50 μg⁄mL endothelial cell growth supplement(ECGS; BD, USA) and 50 μg⁄mL heparin (GUOAO,Shanghai, China). Cultures were maintained at 37 °C ina humidified 5%CO2 incubator. HUVECs were identifiedby their typical cobblestone morphology under micro-scope. Cells were cultured in Medium199 (M199;Hyclone) with supplement as described since the secondpassage. HUVECs from 3 to 5 passages were used forexperiments.

Cell Treatment Protocol

To ascertain the appropriate concentration of ox-LDL to induceHUVECs oxidative injury, we treated cells with different

concentrations of ox-LDL (Yiyuan Biotechnologies, Guangz-hou, China) for 24 h. To explore the effect of resveratrol on ox-LDL-induced cell injury, cells were incubated with differentconcentrations of RESV (2, 10, 50 μmol/L) in the presence ofox-LDL (100 mg/L) for 24 h. In separate experiments, weadded resveratrol to the culture 30 min before ox-LDLstimulation to find out whether pretreatment had an extraeffect. In subsequent experiments, we added 3-methyladenine (3-MA) and 6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide (EX527) respectively intomedia containing both ox-LDL and RESV to explorethe possible mechanisms. Autophagy inhibitor 3-MAand silencing information regulator1 (Sirt1) inhibitorEX527 were purchased from Sigma (St Louis, USA).

Cell Viability Assay

Cell viability was assessed using methyl thiazolyl tetra-zolium (MTT; Sigma) assay according to the manufac-ture recommendation. In brief, HUVECs (1 × 104

cells/well) were plated onto 96-well plates and treatedwith drug-containing media for 24 h. MTT (20 μL,5 g/L) reagent was added to media for 4 h incubation.Then, the supernatant was removed and dimethylsulfox-ide was added to solubilize the formazan crystals. Ab-sorbance was measured at 560 nm with an enzyme-linked immunosorbent assay plate reader (Thermo, Bos-ton, Massachusetts, USA).

Measurement of SOD Levels in Cell Cultures

Superoxide dismutase (SOD) test kit was obtained fromNanjing Jiancheng Bioengineering Institute. HUVECswere seeded in 24-well plates at 3×104 cells/well. Cellswere incubated with assay media for 24 h. The protocolwas according to the manufacturer’s instructions.

Western Blot Analysis

Cells were seeded in 10 cm diameter dishes and treatedwith assay media for 24 h. Then, cells were harvestedand resuspended in ice-cold cell lysis solution. Totalprotein in the supernatant was measured using bicincho-ninic acid (BCA) protein assay kit (Kangcheng BioTech,Shanghai, China). Lysates (80 μg) taken from eachsample were separated by 12%SDS-PAGE, transferredto PVDF membrane (Millipore), blocked with 5 % drymilk for 1 h at room temperature, and probed overnightwith specific antibodies at 4 °C. The blots were incu-bated with HRP-conjugated anti-IgG for 1 h at roomtemperature, detected on Kodak X-Omat film by en-hanced chemiluminescence (ECL; Applygen, Beijing,China). Quantification of band intensity was carried

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out using Image J software. Antibodies against LC3,P62, SIRT1, p-AMPK (Thr172), AMPK were purchasedfrom Cell Signaling Technology (CST; USA) and usedat the dilution of 1:1000. β-actin was carried out as theloading control.

MDC Staining

Cultures were stained with monodansylcadaverine(MDC, 50 μmol/L) at 37 °C for 40 min. After incuba-tion, cells were washed three times with PBS, fixed with5 % paraformaldehyde, and observed under fluorescencemicroscope (Leica, Germany) immediately.

Transmission Electron Microscope

Cells were fixed with 2.5 % glutaraldehyde in 0.1 mol/LPBS for 60 min at room temperature, and post-fixed in1 % osmium tetroxide for 30 min. We sent samples intothe electron microscope chamber (Zhongshan School ofMedicine, Sun Yat-sen University) to produce sections.The ultrastructure of cells was then studied under atransmission electron microscope (tecnai G2 spiritTWIW,FEI,USA).

Quantitative Real Time PCR for mRNA ExpressionAnalysis

Total RNA was extracted with Trizol reagent (Invitrogen).500 ng of total RNA from each sample were used for cDNAsynthesis using Prime Script RT reagent Kit (TaKaRa).Primers used in the qRT-PCR evaluation were designed:SIRT1(forward):5′-CGTCTTATCCTCTAGTTCTTGTG-3′,SIRT1(reverse):5′-ATCTCCATCAGTCCCAAATCC-3′; β-actin(forward):5′-AACCGCGAGAAGATGACCCAGATCATGTTT-3′, β-actin(reverse):5′-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3′. cDNA was subsequentlyamplified with the SYBR Premix Ex TaqTM Kit(TaKaRa) in 8 Strip PCR tubes using the iQ5 instrument(Bio-Rad) as follows: 30 s at 95 °C for enzyme activa-tion followed by 40 cycles of 5 s at 95 °C, 20 s at60 °C, 1 s at 72 °C for the amplification step, and 30 sat 40 °C for the cooling step. Changes in the expressionof target gene (Sirt1) were measured relative to the meancritical threshold (CT) values of β-actin gene.

Statistical Methods

Data were expressed as the mean ± SD for at least threesets of independent experiments. Statistical comparisonsamong multiple groups were performed by one-wayANOVA followed by LSD test using the SPSS 13.0software. A P<0.05 was considered statistically significant.

Results

Resveratrol Attenuated Decreases in Cell Viability and SODLevel Induced by ox-LDL

Figure 1a shows the morphology of HUEVCs incubatedunder normal growth condition. Ox-LDL decreased cellviability in a dose-dependent way as shown in Fig. 1b.There was a statistical significance in the 100 mg/L treatedgroup (*P<0.05), at 78.2±2.5 % of the control group, so wetook 100 mg/L as a proper dose to induce oxidative damagein subsequent experiments. The data showed that resveratrolattenuated the detrimental effect of ox-LDL on cell viabilityin the 10 μmol/L treated group (#P<0.05 versus the ox-LDL group), at 83.4±1.4 % of the control group while ox-LDL treatment (*P<0.05 versus the control group) at 72.9±1.7 % of the control group (Fig. 1c). The pretreatment groupdid not show any advantage (data not shown). We took10 μmol/L as the regular concentration of resveratrol treat-ment subsequently. The extent of oxidative damage wasmeasured by SOD level as shown in Fig. 1d. The SODconcentration was 16.82±0.27 U/ml in the control group.Ox-LDL caused a significant decrease in the SOD level(14.37±0.21 U/ml; *P<0.05 versus the control group). Ad-dition of resveratrol reversed the decrease in SOD concen-tration exerted by ox-LDL in the 10 μmol/L and 50 μmol/Ltreated group (#P<0.05 versus the ox-LDL group), at16.41±0.27 U/ml and 16.39±0.17 U/ml respectively.

Protective Effect of Resveratrol Accompaniedby Autophagy Upregulation

The function of resveratrol is to induce basal autophagy[12], and it has also been shown to mediate a protectiveeffect, through autophagy, in atherosclerosis [4, 13]. Inour study, we determined the autophagic level inHUVECs among different groups. We found that ox-LDL upregulated the protein level of LC3II/LC3I andP62 (*P<0.05 versus the control group). Moreover, therewas a significant increase (#P<0.05 versus the ox-LDLgroup) in LC3II/LC3I expression compared to a reduc-tion in P62 expression in the ox-LDL combined withresveratrol treatment group (Fig. 2a). These results indi-cated that resveratrol could induce autophagy inHUVECs exposed to ox-LDL. The autophagic vacuoleswere observed under fluorescence microscope by stainingcells with monodansylcadaverine (Fig. 2b). The picturesshowed an accumulation of autophagic vacuoles in cellsboth in the ox-LDL treated group and the ox-LDL com-bined with resveratrol treatment group. To further confirmthe activation of autophagy, we examined ultrastructuralchanges in HUVECs with transmission electron microscope.At the ultrastructural level, autophagosomes are defined as a

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double-membraned structure containing undigested cytoplas-mic contents (white arrows in Fig. 2c). Autolysosomes havea single limiting membrane and contain cytoplasmicmaterials in degradation stage (black arrows inFig. 2c), which are generated by the fusion of autopha-gosomes and lysosomes. The EM images also displayedphagophores (double arrows in Fig. 2c). No autophagicvacuole was found in the control group by MDC stain-ing and EM.

Resveratrol Increased the mRNA Level and ProteinExpression of Sirt1

Resveratrol has been known as a sirtuin activator whichmimics caloric restriction in lifespan research [10, 12].To understand the mechanisms involved, we evaluatedthe expression of Sirt1 by RT-PCR and Western blot.

Resveratrol increased the mRNA level of Sirt1 in a dosedependent manner in HUVECs exposed to ox-LDL(Fig. 3a). This result was in parallel with the proteinexpression among different groups as shown in Fig. 3b(*P<0.05 versus the control group, #P<0.05 versus theox-LDL combined with 10 μmol/L RESV treatmentgroup). Resveratrol alone did not significantly alter theSirt1 expression versus the control group.

3-MA Blocked the Protective Effect of Resveratrolby Inhibiting Autophagy

To further investigate the role of autophagy in the pro-tective effect of resveratrol, we measured whether 3-MA,an autophagy inhibitor, could affect the cell viabilityreversed by resveratrol. The cell viability in the ox-LDL treated group, the ox-LDL combined with RESV

Fig. 1 Effects of resveratrol on cell viability and SOD level inHUVECs treated with ox-LDL. a The morphology of HUVECs undernormal culture condition (200×); b Cell viability of HUVECs treatedwith different concentrations of ox-LDL; c Cell viability of HUVECs

treated with different concentrations of RESV in the presence of ox-LDL(100 mg/L); d SOD level of HUVECs in different groups. *P<0.05 versus the control group, #P<0.05 versus the ox-LDL group(treated with 100 mg/L ox-LDL alone), n=5

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treatment group and the 3-MA (3 mmol/L) additionalgroup was at 84.4±1.9 %, 91.4±3.3 % and 80.4±2.7 %of the control group respectively (*P<0.05 versus thecontrol group, #P<0.05 versus the ox-LDL group, △P<0.05 versus the ox-LDL combined with RESV treatmentgroup). The MTT assay indicated that 3-MA abolishedthe protective effect of resveratrol exerted on HUVECswith ox-LDL exposure (Fig. 4a). The protein expressionof LC3II/LC3I and P62 confirmed the inhibition of

autophagy by 3-MA (Fig. 4c). The mRNA level(Fig. 4b) and protein expression (Fig. 4c) of Sirt1 werenot affected by addition of 3-MA. We detected the ex-pression of AMPK-an important protein in autophagyregulation. In Fig. 4c, we found that the phosphorylationlevel of Thr172 in the active site of the AMPK catalyticsubunit (p-AMPK) was upregulated by resveratrol treat-ment and was not influenced by addition of 3-MA. Insummary, autophagy played an important role in the

Fig. 2 The protective effect of resveratrol accompanied by autoupha-gic level upregulation. a Protein expression of LC3II/LC3I and P62 indifferent groups. *P<0.05 versus the control group, #P<0.05 versusthe ox-LDL group, n=3. b MDC staining of HUVECs under fluores-cence microscope (400×). c Electron microscope analysis of HUVECs.

White arrows indicate autophagosomes. Black arrows indicate auto-phagolysosome and double arrows indicate phagophores. In b and c,cells were treated with normal medium (control), ox-LDL (100 mg/L)and ox-LDL combine with RESV (10 μmol/L) respectively

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protective effect of resveratrol on oxidative damage inHUVECs. Additionally, Sirt1 and AMPK could act up-stream of autophagy regulation pathway.

Resveratrol Protected HUVECs from ox-LDL-InducedInjury via Sirt1-Dependent Autophagy

In this part, we studied whether resveratrol could protectHUVECs from ox-LDL-induced injury via Sirt1-dependentpathway. We added EX527 (10 μmol/L), the inhibitor ofSirt1, into the culture medium. As Fig. 5a shows, cellviability in the ox-LDL treated group, the ox-LDL com-bined with RESV treatment group and the EX527 additionalgroup was at 79.1±3.4 %, 91.3±3.8 % and 73.7±1.1 % ofthe control group respectively (*P<0.05 versus the controlgroup, #P<0.05 versus the ox-LDL group, △P<0.05 versusthe ox-LDL combined with RESV treatment group). Theprotective effect of resveratrol on cell viability of HUVECstreated with ox-LDL was abolished by EX527. The mRNAlevel of Sirt1 decreased significantly (*P<0.05 versus thecontrol group, #P<0.05 versus the ox-LDL group) by addi-tion of EX527 (Fig. 5b). The protein expression of Sirt1 wasin accordance with the mRNA levels (Fig. 5c). The proteinexpression of LC3II/LC3I was down-regulated while P62was upregulated by addition of EX527 (Fig. 5c), whichindicated an inhibition of autophagy. These data suggestedthat resveratrol protected HUVECs from ox-LDL-inducedinjury via Sirt1-dependent autophagy. However, there wasno significant change in the ratio of p-AMPK to AMPK inthe EX527 additional group (Fig. 5c), suggesting thatAMPK could act upstream of the AMPK/Sirt1 pathway inautophagy regulation.

Discussion

In the present study, we provide evidence suggesting thatresveratrol could protect HUVECs from ox-LDL inducedoxidative damage through the upregulation of autophagy. Inparticular, our study demonstrates that: 1) resveratrol attenu-ates the detrimental effect of ox-LDL on the viability ofHUVECs and oxidative stress; 2) the protective effects ofresveratrol on the vasculature are mediated in part byautophagy; and 3) resveratrol-induced autophagy is activat-ed through the AMPK/SIRT1 pathway.

Risk of coronary heart disease and myocardial infarctionare strongly associated with ox-LDL particles [2]. The en-dothelium is highly sensitive to oxidative injury caused byox-LDL. Therefore, in this study, ox-LDL was used indesigning an oxidative stress model in HUVECs.

Resveratrol has protective potentials against multiple tar-gets related to cardiovascular diseases. In particular, resvera-trol has an intrinsic anti-oxidant capacity that could be relatedto its chemopreventive effects [7]. To evaluate the effect ofresveratrol on ox-LDL-induced HUVECs, we assessed cellviability and SOD levels, a general indicator of antioxidantability. We found that resveratrol reversed decreases in viabil-ity and SOD levels that had been induced by ox-LDL. Resver-atrol maintained cell viability and inhibits oxidation. Thefindings from our study reveal, for the first time, a protectiveeffect of resveratrol in ox-LDL-treated HUVECs. This effectis, in part, associated with the anti-oxidant effects of resvera-trol. Therefore, we believe that resveratrol could potentially beused in the treatment of atherosclerosis.

As already indicated, removal of LDL is important for thetreatment of oxidation and cell damage. Macroautophagy,

Fig. 3 Resveratrol increased the mRNA level and protein expressionof Sirt1. amRNA level of Sirt1 in different groups. *P<0.05 versus thecontrol group, #P<0.05 versus the ox-LDL combined with 10 μmol/L

RESV treatment group, n=5. b Protein expression of Sirt1 in differentgroups. *P<0.05 versus the control group, #P<0.05 versus the ox-LDL combined with 10 μmol/L RESV treatment group, n=3

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hereafter referred to as autophagy, is a phylogeneticallyconserved catabolic pathway that delivers cytoplasmic com-ponents, such as damaged organelles or long-lived proteins,to lysosomes for bulk degradation [12]. Oxidative stress is awell-known stimulus of autophagy to facilitate the removalof damaged material. Resveratrol has been widely known asa sirtuin activator inducing basal autophagy, with cardiovas-cular benefits and lifespan extension [9, 11, 12, 14]. LC3and P62 are two well-known markers of autophagy. Anenhanced ratio of LC3II/LC3I is a marker of an increase inautophagosomes while P62 expression is inversely correlat-ed with autophagic flux [15, 16]. In this study, we found that

ox-LDL upregulated the LC3II/LC3I protein ratio, and in-creased P62 levels, which indicated an accumulation inautophagosomes rather than an increase in autophagic flux.However, the protective effect of resveratrol was accompa-nied by LC3II accumulation and P62 degradation, therebyindicating upregulation of autophagy. The EM images andMDC staining revealed autophagosomes, confirming theactivation of autophagy. Although it has been considered acell death pathway, recent evidence suggests that autophagyis mostly a cytoprotective mechanism that allows cells tomobilize their energy reserves and to recycle damagedorganelles under conditions of oxidative stress [12].

Fig. 4 3-MA blocked the protective effect of resveratrol by inhibitingautophagy. a Cell viability of HUVECs treated with 3-MA additionalculture medium. *P<0.05 versus the control group, #P<0.05 versusthe ox-LDL only group, △P<0.05 versus the ox-LDL combined withRESV treatment group. b mRNA level of Sirt1 in different groups. *P

<0.05 versus the control group. c Protein expression of LC3II/LC3I,P62, Sirt1 and p-AMPK/AMPK in different groups. *P<0.05 versusthe control group, #P<0.05 versus the ox-LDL only group, △P<0.05versus the ox-LDL combined with RESV treatment group

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However, if autophagy is not engaged as part of the oxida-tive stress response in atherosclerotic plaques or excessiveoxidative damage overcomes the cellular defenses, cellsprobably die via apoptosis. So, the role of autophagy inthe protective effect of resveratrol on ox-LDL induced ox-idative damage remains unclear. We added 3-MA, anautophagy inhibitor, into the medium and found that theprotective effect of resveratrol was abolished by its addition.Our study results suggest that moderate autophagy inducedby resveratrol could be one of the potential mechanismsunderlying the beneficial effects of resveratrol as a therapyfor cardiovascular disease.

Sirt1, a NAD-dependent histone deacetylase, is an essentialmediator of longevity in normal cells by calorie restriction [10,17]. Sirt1 has been shown to increase autophagy flux throughthe deacetylation of essential autophagic factors, such as ATG5and ATG7 [12]. Resveratrol, known as an indirect Sirt1 acti-vator, could not only induce autophagy but also improvesendothelial dysfunction [18–20]. In our current study, wefound that the level of Sirt1 protein was increased by resvera-trol in a dose-dependent manner. Moreover, the mRNA levelsof Sirt1 were upregulated in parallel with protein expression.These results imply that Sirt1 may function as a commontrigger for the activation of autophagy by resveratrol.

Fig. 5 Resveratrol protected HUVECs from ox-LDL-induced injuryvia Sirt1-dependent autophagy. a Cell viability of HUVECs treatedwith EX527 additional culture medium. *P<0.05 versus the controlgroup, #P<0.05 versus the ox-LDL only group, △P<0.05 versus theox-LDL combined with RESV treatment group. bmRNA level of Sirt1

in different groups. *P<0.05 versus the control group. c Proteinexpression of LC3II/LC3I, P62, Sirt1 and p-AMPK/AMPK in differentgroups. *P<0.05 versus the control group, #P<0.05 versus the ox-LDL only group, △P<0.05 versus the ox-LDL combined with RESVtreatment group

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To investigate the mechanism of resveratrol-inducedautophagy, we examined the activation of AMPK, asensor of energy molecules. Resveratrol is known toactivate AMPK, possibly via inhibition of mitochondrialATP synthesis and cAMP phosphodiesterase [19, 21, 22].Sirt1 is activated downstream, possibly because AMPKactivation increases the NAD+/NADH ratio, with Sirt1known to be NAD+-dependent [23, 24]. It has beenreported that Sirt1 contributes to AMPK activationthrough deacetylation of the upstream kinase, LKB1[25]. In our study, 3-MA did not affect the expressionof Sirt1 and AMPK, both of which are crucial forautophagy regulation. As AMPK and Sirt1 signaling arestrongly intertwined, we used the Sirt1 inhibitor EX527to determine the relationship between Sirt1 and AMPKin resveratrol-induced autophagy regulation.

The addition of EX527 did not affect the expressionof AMPK, indicating that AMPK may act upstream ofSirt1 in the autophagic regulation of endothelial cells.Resveratrol activates AMPK in vitro which acts upstreamof Sirt1, suggesting that resveratrol activated Sirt1 indi-rectly. There are also findings that demonstrate thatresveratrol activates Sirt1 to deacetylate fluorophore-tagged substrates rather than native substrates in vitro[24, 25]. Additionally, EX527 abolishes the protectiveeffects that are a result of inhibiting autophagy. Thisfinding revealed the crucial role of Sirt1 in resveratrolexerting its protective effects, through the induction ofautophagy.

In summary, our data suggest a protective effect ofresveratrol on oxidative damage in HUVECs that hadbeen exposed to ox-LDL. Our findings indicate the po-tential resveratrol possesses for preventing and treatingatherosclerosis. We also demonstrated a novel favorablerole of autophagy in this effect, but how to turn on theprotective effects of autophagy without activating un-wanted death pathway will be both a promising strategyand a challenge for clinicians. Moreover, it remains un-clear what happens with a large amount of oxidizedlipids in the cytoplasm of foam cell undergoingautophagy.

The exact dose of resveratrol to be used in the humanbody to induce moderate autophagy has not yet been ana-lyzed in full detail. To investigate a safe resveratrol dose toinduce moderate autophagy, more clinical trials need to bedone. The AMPK/SIRT1 pathway involved in resveratrol-induced autophagy regulation may provide some clueswhen searching for new drugs that can be applied to endo-thelial protection.

Acknowledgments This work was supported by National NaturalScience Foundation of China (30973843) and National Natural ScienceFoundation of China (30870987).

Conflict of interest None declared

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