Research Article SIRT1 Inhibition Affects Angiogenic...

Research ArticleSIRT1 Inhibition Affects Angiogenic Properties of Human MSCs

Botti Chiara12 Caiafa Ilaria1 Coppola Antonietta1 Cuomo Francesca1

Miceli Marco3 Altucci Lucia13 and Cobellis Gilda12

1 Department of Biochemistry Biophysics and General Pathology Second University of Napoli Via L De Crecchio 780138 Napoli Italy

2 Istituto Nazionale Tumori Struttura Complessa Oncologia Medica Melanoma Immunoterapia Oncologica e Terapia InnovativaVia Mariano Semmola 80131 Napoli Italy

3 Institute of Genetics and Biophysics ldquoA Buzzati-Traversordquo CNR Via P Castellino 111 80131 Napoli Italy

Correspondence should be addressed to Botti Chiara chiara bottiyahooit and Cobellis Gilda gcobellisunina2it

Received 18 July 2014 Revised 7 August 2014 Accepted 8 August 2014 Published 27 August 2014

Academic Editor Zongjin Li

Copyright copy 2014 Botti Chiara et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Humanmesenchymal stem cells (hMSCs) are attractive for clinical and experimental purposes due to their capability of self-renewaland of differentiating into several cell types Autologous hMSCs transplantation has been proven to induce therapeutic angiogenesisin ischemic disorders However the molecular mechanisms underlying these effects remain unclear A recent report has connectedMSCsmultipotency to sirtuin families showing that SIRT1 can regulate MSCs function Furthermore SIRT1 is a critical modulatorof endothelial angiogenic functions Here we described the generation of an immortalized human mesenchymal bone marrow-derived cell line and we investigated the angiogenic phenotype of our cellular model by inhibiting SIRT1 by both the genetic andpharmacological level We first assessed the expression of SIRT1 in hMSCs under basal and hypoxic conditions at both RNA andprotein level Inhibition of SIRT1 by sirtinol a cell-permeable inhibitor or by specific sh-RNA resulted in an increase of premature-senescence phenotype a reduction of proliferation rate with increased apoptosis Furthermore we observed a consistent reductionof tubule-like formation and migration and we found that SIRT1 inhibition reduced the hypoxia induced accumulation of HIF-1120572protein and its transcriptional activity in hMSCs Our findings identify SIRT1 as regulator of hypoxia-induced response in hMSCsandmay contribute to the development of new therapeutic strategies to improve regenerative properties of mesenchymal stem cellsin ischemic disorders through SIRT1 modulation

1 Introduction

Human mesenchymal stem cells (hMSCs) have become animportant tool for cell-based strategiesThey can differentiateinto a variety of cell types such as muscle neural precursorscardiomyocytes and perivascular cells and are currentlybeing tested in several approved clinical trials [1] hMSCscan improve myocardial remodeling in infarcted heart [2] orpromote angiogenesis in critical limb ischemia [3] due totheir capacity to stimulate endothelial progenitor cells Fur-thermore hMSCs support neoangiogenesis also by releasingsoluble factors that stimulate angiogenesis [4ndash9] Howeverthe molecular mechanisms of beneficial effects from hMSCs-based therapy remain unclear

A recent report showed that SIRT1 might regulate MSCsfunction providing a connection between sirtuin familiesand MSCs multipotency [10]

Sirtuins are classified as class III histone deacetylases(HDACs) [11] originally identified in yeast They modulatea wide range of biological processes spanning from DNArepair and oxidative stress responses to energy metabolismSirtuins activity is controlled by the cellular [NAD+][NADH] ratio where NAD+ works as an activator whereasnicotinamide and NADH act as inhibitors

In mammals the sirtuin family comprises seven mem-bers (SIRT1ndashSIRT7) with different biological functions andsubcellular localizations [12ndash14] SIRT1 SIRT6 and SIRT7are mainly nuclear whereas SIRT2 is found primarily in the

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 783459 12 pageshttpdxdoiorg1011552014783459

2 BioMed Research International

cytosol SIRT3 SIRT4 and SIRT5 are mitochondrial proteins[12] Sirtuins are generally known to regulate the acetylationlevels and the activity of histone and nonhistone regulatoryproteins

To date sirtuins have emerged as potential therapeutictargets for treatment of human pathologies such as cardio-vascular disease inflammation and cancer [15] SIRT1 is themost studied member of sirtuins It acts in various cellularprocesses and exerts its action activating and deactivatingfactors such as NF-120581B p53 p73 SOD and hypoxia-inducibletranscription factors (HIFs) [16ndash19]

Since SIRT1 targets several proteins in distinct signalingpathways modulation of SIRT1 activity alters the biologicalactivity of entire signaling networks modifying disease pro-gression such as pathological angiogenesis or atherosclerosis

An important component of pathological angiogenesis isrepresented by hypoxia that alters the cellular redox state andactivates SIRT1 In addition hypoxia works through multiplepathways to regulate angiogenesis for instance through themodulation of secreted angiogenic proteins such as vascularendothelial growth factor (VEGF) stimulated by increasedexpression of transcription factors such as HIF-1120572 [20]

Furthermore a ldquoprotectiverdquo role of SIRT1 in endothelialcells was described [21 22] A recent report has investigatedthe function of SIRT1 in regulating the differentiation ofmesenchymal stem cells by deacetylating 120573-catenin in mice[10] Several studies demonstrated that inhibition of SIRT1impairs cell growth in cancer cells [23 24] Gorenne et al[25] reported that SIRT1 expression was reduced in humanatherosclerotic plaques and in vascular smooth muscle cellsHowever to the best of our knowledge the effects of SIRT1modulation on angiogenesis in hMSCs have not been studiedyet

In this study we described the generation of an immor-talized human mesenchymal bone marrow-derived cell line(MeBM) and we investigated whether SIRT1 has an effect onangiogenic capability of hMSCs by inhibiting SIRT1 throughpharmacological and genetic approaches

Since recent studies have identified SIRT1 as a criticalmodulator of angiogenesis [18 26] we tested whether theinhibition of SIRT1 activity is associated with the reduction ofangiogenic ability of hMSCs and impaired hypoxic responsein these settings We found that the inhibition of SIRT1 activ-ity resulted in reduced capacity to proliferate to migrate andto form three-dimensional networks of vessel-like structuresIn addition SIRT1 inhibition reduced the hypoxia-inducedaccumulation of HIF-1120572 and its transcriptional activity inhMSCs

Our results suggested that SIRT1 is involved in angiogenicresponse of hMSCs in vitro and modulates the hypoxicresponse through inhibiting HIF-1120572 activity

Our findings may help to understand the role of SIRT1 inhMSCs in promoting angiogenesis and may contribute to thedevelopment of new strategies to improve the hMSCs-basedregenerative effects by modulating SIRT1 activity

2 Methods

21 Reagents Sirtinol was purchased from Selleck ChemicalsLLC (Houston TX USA) Culture medium and its supple-ments including antibiotics and fetal bovine serum (FBS)were purchased from Euroclone (Italy) Primary antibodiesagainst SIRT1 (Abcam Cambridge UK) HIF-1120572 (Santa CruzBiotechnology Santa Cruz CA USA) and tubulin (Sigma-Aldrich Milan Italy) were used Sirtinol was dissolved indimethyl sulfoxide (DMSO Sigma-Aldrich) to the appropri-ate concentrations according to reported procedures DMSOwas also present in the corresponding control

22 Cell Lines and Culture Medium Human mesenchymalstem cells (hMSCs) were obtained from bone marrow asdescribed by Cobellis et al [8] Cells were plated in RPMI1640 growth medium (Euroclone SPA Italy) containing 10heat-inactivated FBS 1 Pen-strep and 1 L-Glutamine

Cells were maintained as monolayers in a humidifiedatmosphere containing 5 CO

2at 37∘C and the culture

medium was replaced every two daysHypoxic culture conditionswere achieved in aBDGasPak

EZAnaerobeGasGenerating Pouch System (BDBiosciencesSan Diego) As certified by the manufacturer the AnaerobeGas Generating Pouch System produces an atmospherecontaining 10 carbon dioxide and 1 oxygen

Starvation conditions were obtained incubating cells inRPMI 1640 containing 02 FBS

23 Infection After plating bone marrow cells were grownto confluence and coinfected with HPV16 E6E7 and hTERTlentiviral vectors (infection number 1) After a week thecells were split and infected again only with hTERT (infec-tion number 2) and cultured until stabilization Sampleswere observed and photographed with DMI 6000 invertedmicroscope (Leica Microsystems) using Leica LAS ImageAnalysis software (Leica Microsystems) The protocol wasalso reported in Miceli et al [27]

24 hPV16 E6E7 and hTERT Lentiviral Production HIV-1based SIN lentiviral vectors were derived from SINF-MU3-W-S vector backbone hPV16 E6E7 was inserted upstreamof an encephalomyocarditis virus internal ribosome entrysite- (IRES-) yellow fluorescent protein (YFP) gene cassetteinto SINF-MU3-W-S to generate SINF-MU3-E6E7-IRES-YFPW-S SINF-MU3-hTERT-IRES-GFPW-S was generatedby inserting hTERT cDNAupstream of an IRES-green fluore-scent protein (GFP) gene cassette into SINF-MU3-W-S VSV-G-pseudotyped lentiviral vectors were generated in 150mmtissue culture dishes by transient cotransfectionwith (1) VSV-G-expressing construct pCMV-VSV-G (Invitrogen USA)(66 120583g) (2) packaging construct pCMVΔR82 (addgene)(48 120583g) and (3) lentiviral vector plasmids (pSin hTERT orPsin E6-E7) (66 120583g) into subconfluent HEK 293FT cells(Invitrogen) by calcium phosphate precipitation (ClontechCalphos Mammalian Transfection Kit) The supernatantcontaining the virus was produced in HEK-293FT collectedfiltered and used to infect bone marrow cells

BioMed Research International 3

25 Gene KnockdownUsing Lentiviral Vector Cells (105) weregrown in RPMI 1640 medium 45 gL glucose (EurocloneSPA Italy) supplemented with 20 FBS (Euroclone SPAItaly) 100UmL Pen-strep (Lonza Group Ltd) and 2mML-Glutamine (Lonza Group Ltd) at 37∘C in 5 CO

2fully

humidified atmosphere Cells were first grown for 24 h andthen infected with the rLVH1sh2Sirt1EF1GFP Lentiviruswith a 25MOI overnight as described by Miceli et al [27]

26 RNA Extraction and qPCR Total RNAwas isolated fromhMSCs by miRNeasy Mini kit (QIAGEN GE) 500 ng wasconverted to cDNA using the Quantitect Reverse Transcrip-tion kit (QIAGEN GE)

qPCR assays were performed using an iCycler (BioRadLaboratories USA) and the Sybergreen Super mix (BioRadLaboratories USA) The primer sequences and qPCR condi-tions are available on request

27 Protein Extraction and Western Blot Analysis hMSCswere incubated with 100 120583M sirtinol for 24 h and then wecollected lysates from cells exposed to 1 O

2for 6 h

Cells were lysed in buffer containing 20mM Tris HCl100mM NaCl 10mM MgCl

2 1 NP40 10 glycerol 01M

NaF 100 120583M sodium vanadate and protease inhibitors mix-ture (Roche LTD GE) Equal amounts of supernatant wereseparated by SDS-polyacrylamide gels Proteins were trans-ferred to nitrocellulose membranes (WhatmanProtran GEHealthcare) and membranes were blocked with blockingbuffer (TBS-Tween buffer containing 5milk) Subsequentlythe membranes were incubated with primary antibodiesat 4∘C overnight After three washes for 101015840 with TTBSbuffer (50mM Tris HCl pH 8 150mM NaCl and 05Tween-20) the membranes were incubated with horseradishperoxidase-conjugated anti-mouse or anti-rabbit antibody(1 10000 Santa Cruz Biotechnology Santa Cruz CA USA)for 1 h at room temperature and then washed for 30min withTTBS bufferThe resulting immunoblots were detected usingAmersham ECL Plus (GE Healthcare)

28 Senescence Associated Beta-Galactosidase (SA-Beta-Gal)Staining Cells were cultured on 6-well plates at a densityallowing reaching 20ndash30 confluence and exposed for 24 h to50 and 100 120583M sirtinol After exposure the cells were washedthree timeswith inhibitor-freemediumand cultured for up toadditional 8 days On the ninth day the cells were fixed with2 (vv) formaldehyde02 (vv) glutaraldehyde for 10minThe cells were then washed twice with PBS and incubatedwith staining solution (30mM citric acidphosphate buffer(pH 6) 5mM K

4Fe(CN)

6 5mM K

3Fe(CN)

6 150mMNaCl

2mMMgCl2 and 1mgmL X-Gal solution (all reagents were

purchased from Sigma Milan Italy)) at 37∘C for 24 h Thecells were photographed and quantified with an invertedmicroscope (Leica Heidelberg Germany)

29 Cell Proliferation Cells were plated (5 times 103 cellswellin 96 well plates) in RPMI 1640 (Euroclone SPA Italy) andallowed to attach overnight The day after hMSCs weretreated with 100 120583M sirtinol for 24 48 and 72 hThe number

of living cells was measured by determination of ATP cellularlevels using ViaLight Plus Kit (Lonza Group Ltd) The kit isbased upon the bioluminescent measurement of ATP that ispresent in all metabolically active cells The bioluminescentmethod utilizes an enzyme luciferase which catalyzes theformation of light from ATP and luciferin The emitted lightintensity is linearly related to the ATP concentration and ismeasured using a luminometer RLUs (relative light units) areinternal unit of the kit proportional to the amount of lightproduced for ATP unit

All experiments were performed in triplicates

210 Flow Cytometry Analysis Cells were treated with100 120583Msirtinol for 24 48 and 72 h Cells were resuspended inthe staining solution containing RNAseA propidium iodide(50 120583gmL) sodium citrate (01) and NP40 (01) in PBS1X for 30min in the dark Cell cycle distribution was assessedwith a FACScalibur flow cytometer (Becton Dickinson SanJose CA USA) and 10000 cells were analyzed by ModFitversion 3 Technology (Verity Software House Topsham MEUSA) and Cell Quest (Becton Dickinson San Jose CA USA)[27]

211 Migration Assay Cells were plated on 24-well plates ata density allowing reaching 50ndash75 confluence and hMSCswere treated for 24 48 and 72 h with 100 120583M sirtinol A totalof 15 times 104 cells were resuspended in 250120583L of RPMI 1640containing 02 FBS and pipetted in the upper chamber ofa modified Boyden chamber (Costar Transwell assay 8120583mpore size Corning NY) The chamber was placed in a 24-well culture dish containing 750120583L complete RPMI 1640with 10 FBS and growth factors After 24 h incubationat 37∘C transmigrated cells were counted by independentinvestigators at the inverted microscope

212 Capillary Tube Formation Assay in Matrigel Cells wereplated on 6-well plates at a density allowing reaching 50ndash75 confluence and hMSCs were treated for 24 h with 50and 100 120583M sirtinol For analysis of capillary tube formation150 120583L Matrigel (Becton Dickinson San Jose CA USA) waslaid into a 96-well plates (BD Falcon Heidelberg Germany)and incubated at 37∘C for 30 minutes Cells were trypsinizedand 3 times 104 cells were suspended in 150120583L of mediumand plated onto Matrigel Cells were incubated at 37∘C andcapillary tube formation in Matrigel was observed under aninverted microscope (Leica Heidelberg Germany) after 4and 24 h of incubation

213 Statistical Analysis All data are represented as meanplusmn SD Statistical significance was evaluated by performingStudentrsquos 119905-test and significance was accepted if 119875 value waslt005

3 Results

31 Immortalization of hMSCs The ectopic expression ofhTERT has been reported to extend the life span of cells [28]


However the use of hTERT alone is not sufficient to immor-talize hMSCs requiring the combinatorial expression ofhuman papillomavirus type 16 genes (HPV16) E6 and E7 [29]

Therefore primary hMSCswere infectedwithHPV16 E6-E7 and hTERT lentiviral vectors expressing pSin hTERT andpSin E6-E7 [30] using a multi-infection program as reportedin Methods section Two clones were obtained and testedfor the presence of hTERT and E6-E7 transcripts Based onRT-PCR data both clones (MeBM1E1 MeBM1E2) showedsimilar levels of hTERT and E6-E7 transcripts No hTERTand E6-E7 expression were detected in untransduced hMSCs(Supplementary Figure 1(a) see the Supplementary Mate-rial available online at httpdxdoiorg1011552014783459)The resulting cell linesmaintained a fibroblast-like phenotypecomparable to primary hMSCs and showed no differences inhMSCsmarkers expression such as CD73 CD90 and CD105(Supplementary Figure 1(b)) Thus these immortalized mes-enchymal cells (MeBM1E1 MeBM1E2) represent a valuablemodel that can be used for basic studies of mesenchymalbiology

32 Differential Sirtuin Expression in hMSCs Using theMeBM1E1 clone we assessed the expression profile of theSirt1ndashSirt7 genes We collected RNAs from cells exposed toeither 21 O

2or 1 O

2for 24 h and we performed RT-qPCR

analysis to quantify their expression As shown in Figure 1(a)a significant induction of Sirt1 and Sirt7 (119875 value le 005) wasdetected at mRNA levels in hMSCs under hypoxic conditionscompared to normoxia No significant differences in othersirtuin transcripts were observed under the same conditions

Next we evaluated the contribution of hypoxia on SIRT1protein expression andwe collected lysates fromcells exposedto 1O

2for 24 h in the presence or absence of growth factors

(ie serum)As shown in Figure 1(b) Western blot analysis showed

that there was no change in SIRT1 protein levels in hMSCsexposed to 1 O

2for 24 h grown in presence of serum

whereas hypoxia increased SIRT1 levels when hMSCs werecultured in low serum conditions These data showed thathypoxia alone did not stimulate SIRT1 protein accumulationwhereas depletion of growth factors in combination withhypoxia resulted in an increase of SIRT1 protein expression

33 Inhibition of SIRT1 Induces Premature Senescence-LikePhenotype in hMSCs To evaluate the effects of targetingSIRT1 we decided to use pharmacological and geneticapproaches to inhibit SIRT1 Genetic inhibition was obtainedby silencing SIRT1 with lentiviral vector expressing sh-Sirt1-GFP In order to determine the infection efficiency greenfluorescent protein (GFP) was monitored using fluorescencemicroscopy after 10 days from infection As shown in Supple-mentary Figure 2(a) high levels of GFP in sh-Sirt1-hMSCswere observedwith a concomitant reduction of SIRT1 protein(Supplementary Figure 2(b)) In addition pharmacologicalinhibition was obtained by sirtinol a cell permeable specificinhibitor of SIRT deacetylase activity [19]

To investigate whether SIRT1 modulates prematuresenescence-like phenotype in hMSCs we examined the effect

of SIRT1 inhibition in our cells hMSCs were treated withsirtinol at 50 and 100 120583M for 24 h After exposure thecells were washed with inhibitor-free medium and culturedfor additional 8 days As shown in Figure 1(c) sirtinolinduced senescence-like morphological changes in hMSCsthat showed enlarged and flattened shapes with a concomi-tant cell number reduction Then we evaluated SA-120573-galactivity a characteristic feature of senescence phenotypeSirtinol increased SA-120573-gal activity in hMSCs compared tocontrol Importantly sirtinol increased SA-120573-gal activity in adose-dependent manner (Figures 1(d)-1(e)) To confirm theprosenescence role of SIRT1 inhibition we examined SA-120573-gal activity in sh-Sirt1 infected cells As expected similarresults were obtained in sh-Sirt1 cells (data not shown)These results demonstrated that inhibition of SIRT1 induceda senescence phenotype in hMSCs

34 Effects of SIRT1 Inhibition on Proliferation We thenexamined the effects of SIRT1 inhibition on the proliferationof hMSCs Cells were treated as previously described andproliferation was measured by intracellular levels of ATPat different times (24 48 and 72 h) Sirtinol significantlyinhibited the proliferation of hMSCs in a time-dependentmanner compared to control Similar results were obtainedin sh-Sirt1 hMSCs (Figures 2(a)-2(b))

To investigate whether inhibition of SIRT1 inducedgrowth arrest or cell death a cell cycle analysis was performedon hMSCs treated with sirtinol for 24 48 and 72 h byfluorescence activated cell sorting (FACS) By this analysiswe observed a significant cell accumulation in pre-G1 phasecorresponding to apoptotic cells after 24 48 and 72 h oftreatment compared to untreated cells No significant effectwas observed in G1 G2 and S phase (Figure 2(c)) comparedto control Similar results were obtained in sh-Sirt1 cells (datanot shown) These data suggested that inhibition of SIRT1obtained by both pharmacological and genetic approachesinduced apoptosis in hMSCs without altering the cell cycledistribution of the cells

35 SIRT1 Inhibition Impairs Migration and Capillary TubeNetwork Formation Then we examined whether inhibitionof SIRT1 impinged on the migration ability of hMSCs Cellswere exposed for 24 48 and 72 h to 100 120583M sirtinol andwe examined the migratory ability of hMSCs in presenceof growth factors The number of migrated cells was sig-nificantly reduced in cells treated with SIRT1 inhibitor in atime-dependent manner (Figure 3(a)) compared to controlConsistent with these findings silenced SIRT1 resulted ina significant reduction of migration compared to control(Figure 3(b))

These results suggested that inhibition of SIRT1 reducesthe capability of hMSCs tomigrate To further assess whetherinhibition of SIRT1 might play a role in the ability to formcapillary-like networks we performed a tubule formationassay We used Matrigel as the basement matrix to inducetubule formation Cells were exposed to sirtinol (50 and100 120583M) for 24 h then sirtinol was removed from the culturemediaThe cells were plated onMatrigel and allowed to form


0

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expr

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SIRT1 SIRT2 SIRT3 SIRT5 SIRT6 SIRT7SIRT4

lowast

NH

lowast

(a)

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SIRT1

Tubulin

(b)

CTR50120583m 50120583M sirtinol 100120583M sirtinol

(c)


(d)

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sirtinolCTR

sirtinol

lowast

lowast

100120583M50120583M

SA-120573

-gal

pos

itive

cells

()

(e)

Figure 1 SIRT1ndashSIRT7 expression in hMSCs and effects of SIRT1 inhibition on phenotype (a) SIRT1ndashSIRT7 mRNA levels were measuredby real-time PCR (RT-PCR) analyses of total RNAs obtained from hMSCs exposed to either 21 O

2(N) or 1 O

2(H) for 24 h and relative

expression (plusmnSD) were shown (b)Western blot analysis of hMSCs grown at either 21O2(N) or 1O

2(H) for 24 h under normal conditions

(upper panel) and serum-starved (lower panel) Antibodies against SIRT1 and tubulin were used (c) Morphological changes in hMSCs wereexamined 8 days after treatmentwith sirtinol (50 and 100 120583M) for 24 h (40xmagnification scale bars = 50 120583m) (d) Representative photographsof blue-stained cells for SA-120573-Gal activity are shown (10xmagnification scale bars = 200 120583m) at 8 days after sirtinol (50 and 100120583M) treatmentcompared to control (CTR) (e) SA-120573-Gal-positive cells were quantified by counting in at least 3 random fields for each condition Resultsshow the mean of three independent experiments Graph represents means plusmn SD 119899 = 3 lowast indicates statistical significance from control119875 le 005

tubule networks in vitro As shown in Figure 3(c) we foundthat hMSCs exposed to 50 120583M sirtinol formed less developedtubule structures than untreated cells within a 4-hour periodand the number of branch points significantly diminished(Figure 3(d)) Additionally cell treatment at a concentrationof 100 120583M sirtinol resulted in the complete suppression of

tubule-like structure formation in contrast to stable tubularnetworks present in the control (Figure 3(c) upper panel-3D) A reduced ability to form tubule-like formation was alsoobserved in sh-Sirt1 cells compared to control (Figure 3(c)lower panel-3D) These data indicated that SIRT1 activity isinvolved in tubule-like formation of hMSCs


0

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24h

100120583M sirtinol CTR 100120583M sirtinol

CTR 100120583M sirtinol

48h

72h

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times104

times104 times104

(a)

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RLU

s

sh-Sirt1

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RLU

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RLU

s

48h24h

72htimes104

times104 times104

(b)

Figure 2 Continued


01

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79525160216

10758120119

50

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0275598146

48

566

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CTR

48h

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ber

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24h

72h

100120583M sirtinol

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

(c)

Figure 2 Effects of SIRT1 inhibition on proliferation cell cycle and apoptosis (a) hMSCs were treated with sirtinol 100 120583M (black bars) orequivalent concentration of DMSO (white bars) for 24 48 and 72 h (b) sh-Sirt1 infected hMSCs were plated and proliferation was measuredfor 24 48 and 72 h Each histogram indicates the RLUs related to cell growth measured at different times Error bars represent SD of119899 = 3 lowast denotes statistical differences when 119875 le 005 is compared to control (c) hMSCs were treated with sirtinol 100 120583M or equivalentconcentration of DMSO for 24 48 and 72 h The cells stained with propidium iodide (PI) were subjected to flow cytometric analysis todetermine the cell distributions at each phase of the cell cycle RLUs are relative light units (RLUs) related to ATP cellular level


0

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CTR

CTR

Mig

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lls (

)

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)

24h 48h

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100120583M sirtinol

lowastlowast

lowast

(a)

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)

lowast

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24h 48h

72h

(b)

Figure 3 Continued


CTR sh-Sirt1200120583m

100120583M sirtinol50120583M sirtinol

(c)

0

5

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Num

ber o

f bra

nch

poin

ts (

)CTR sh-Sirt1100120583M sirtinol50120583M sirtinol

lowast lowast lowast

(d)

Figure 3 Effects of SIRT1 inhibition on migration and capillary tube network formation (a) hMSCs were treated with sirtinol 100 120583M (blackbars) or equivalent concentration of DMSO (white bars) for 24 48 and 72 h (b) sh-Sirt1-hMSCs migration (black bars) compared to control(white bars) at 24 48 and 72 h Each histogram indicates the of migrated cells measured at different times Statistical differences weredenoted with lowast when 119875 le 005 is compared to control (c) Tubule formation promoted by hMSCs treated with sirtinol (50 and 100 120583M)(upper panel) and tubular structures of sh-Sirt1 infected hMSCs plated on Matrigel compared to control (lower panel) Magnification 10xScale bar = 200 120583m (d) Tubule formationwas quantified by counting the number of branch points of the capillary network Data are expressedas mean plusmn SD lowast denotes statistical differences when 119875 le 005 is compared to control

36 Impaired Angiogenic Properties of hMSCs Resulting fromSIRT1 Inhibition Are Mediated by HIF-1120572 Protein To investi-gate whether SIRT1 activity could regulate HIF-1120572 accumula-tion we analyzed the effects of SIRT1 inhibition on HIF-1120572

Cells were incubated with 100 120583M sirtinol for 24 h andthen exposed to 1 O

2for 6 h known to induce HIF-1120572 We

then collected cell lysates and Western blotting analysis wasperformed Interestingly as shown in Figure 4(a) inhibitionof SIRT1 led to a reduction of SIRT1 protein as expected and astrong repression of HIF-1120572 protein accumulation in hypoxicconditions compared to control

To determine if reduction in HIF-1120572 levels conferred bySIRT1 inhibition could affect HIF-1120572 transcriptional activitywe looked at the expression of known HIF-1120572 target genesCells were treated with 100 120583M sirtinol for 24 h and exposedto either 21 O

2or 1 O

2for 6 h Then we collected RNAs

from cells and RT-qPCR analysis was performed As shownin Figure 4(b) the treatment with sirtinol in combinationwith hypoxia significantly reduced the hypoxic induction ofSIRT1 expression and HIF-1120572 target genes Glut1 and VEGFThese data indicated that inhibition of SIRT1 decreased HIF-1120572 protein accumulation and its transcriptional activity underhypoxic conditions

4 Discussion

In the present study we have generated an immortalizedhuman bonemarrow-derivedmesenchymal cell line (MeBM)that represent a valuable tool to study mesenchymal biologyand we investigated whether SIRT1 can influence angiogeniccapacity of these cells

Cell therapywith hMSCs is a promising and safemodalitywith the potential for vascular regeneration in the treatmentof several diseases especially critical limb ischemia Pilothuman studies have shown that autologous transplantationof bone marrow cells induced therapeutic angiogenesis inpatients with critical limb ischemia [3] hMSCs are pluripo-tent progenitors that can differentiate into a variety of celltypes and have been shown to promote angiogenesis both invivo and in vitro It is widely accepted that hMSCs presentin bone marrow are therapeutic cells Emerging evidencesuggests that most of the beneficial effects of hMSCs canbe explained by the secretion of soluble factors that induceendogenous reparatory processes However the mechanismsby which hMSCs promote angiogenesis are not clear

The present investigation was undertaken to identify thefunction if any of SIRT1 in hMSCs and clarify the molecular


SIRT1

Tubulin

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CTR 100120583M

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lowast lowast

lowast

(b)

Figure 4 Effects of sirtinol on SIRT1 expression and HIF-1120572 (a) hMSCs were treated with sirtinol (100120583M) or equivalent concentration ofDMSO (CTR) for 24 h and then exposed to 1 O

2for 6 h All cells lysates were analyzed for SIRT1 HIF-1120572 and tubulin by Western blot

(b) The amounts of SIRT1 VEGF and GLUT1 mRNA of hMSCs treated with sirtinol at concentration of 100 120583M (black bars) or equivalentconcentration of DMSO (white bars) for 24 h and exposed to 1O

2(H) for 6 hwere quantified with real-time PCRThe levels of SIRT1 VEGF

and GLUT1 mRNA of hMSCs exposed to 21 O2(N) for 24 h were measured lowast indicates statistical significance from control 119875 le 005

mechanism by which SIRT1 regulate angiogenesis in hypoxicconditions

Sirtuins represent a family of NAD+-dependent proteindeacetylases involved in several pathologies In mammalsSIRT1 is the most closely related homologue of yeast Sir2 andbelongs to class III histone deacetylases It regulates a widevariety of biological functions including gene expression cellsurvival proliferation differentiation metabolism immuneresponse carcinogenesis and angiogenic response throughmultiple targets [15]

Investigation of sirtuin expression was an importantpreliminary step to study the selective role of class IIIHDACsSince sirtuins have been shown to respond to perturbations inthe ratio of oxidizedNAD+reducedNADHand therefore tomodulate the response to hypoxic stress [31] we first looked atsirtuin expression of the family members both in normoxicand hypoxic conditions

Therefore we assessed the expression profile of the Sirt1ndash7 genes in our cells We found that SIRT1 and SIRT7 weresignificantly upregulated in hypoxic conditions compared tonormoxia

SIRT1 activation improves endothelial function and sup-presses vascular inflammation two central pathophysiolog-ical processes involved in the initiation and progression ofcardiovascular disease [32] In particular SIRT1 has beenshown to protect endothelial cells frompremature senescenceand to regulate angiogenesis and vascular tone [26] thus wedecided to focus our study on SIRT1

In our study we evaluated the contribution of hypoxiaon SIRT1 protein showing that no change in SIRT1 wasdetected in hMSCs grown in presence of growth factorswhereas SIRT1 protein expression increased in hMSCs grownin absence of growth factors under hypoxic conditions Other

experiments will be necessary to clarify this however ourfindings might indicate that SIRT1 may act as a sensor of themetabolic state ofMSCs in stress conditions (ie hypoxia andabsence of growth factors)

Several studies have demonstrated that SIRT1 preventsthe onset of senescence in multiple cell types which exhibitalterations in morphology and gene expression that extin-guish essential cellular functions [31]

It is known that primary cells underwent senescencewhen cultured in vitro showing increased activity of 120573-galactosidase (120573-gal) when assayed at pH 6 [22] Thereforewe decided to evaluate whether inhibition of SIRT1 mightplay a role in undertaking a senescent phenotype in ourcells We found that sirtinol a cell-permeable 2-hydroxy-1-napthaldehyde derivative that acts as a specific and directinhibitor of all NAD+-dependent protein deacetylases ofsirtuin family significantly increased SA-120573-gal activity ina concentration-dependent manner and sustained enlargedand flattened cell morphology

Our results are consistent with previous studies in humanendothelial cells [22 33] mouse fibroblasts [34] and humancancer cells [35] concluding that sirtuins are implicated incellular senescence

It has been demonstrated that senescence alters mes-enchymal stem cell properties such as proliferation andmigration [36] and activity of SIRT1 has been linked to thiscondition [37] In fact when cells undergo senescence theirproliferation declines significantly

Our findings showed that SIRT1 inhibition significantlyinhibited the proliferation rate of hMSCs in a time-dependentmanner and significantly induced apoptosis in hMSCs

Going through our data we also noticed that the effectof genetic inhibition of SIRT1 on angiogenesis response of


hMSCs was less pronounced than pharmacological modu-lation of SIRT1 A possible explanation for this observationis that sirtinol can inhibit other NAD+-dependent proteindeacetylases of sirtuin family especially SIRT2 [22] In con-clusion we suggested that inhibition of SIRT1 both by phar-macological and genetic approaches reduces significantlyangiogenic properties in cultured hMSCs

The connection between sirtuin and HIF proteins iscomplex and the current literature is in part contradictory[19] Recent reports have linked HIF to sirtuin families bydemonstrating that SIRT1 SIRT3 SIRT6 and SIRT7 canregulate the activity of HIF proteins [19 38] Lim et al [18]demonstrated that SIRT1 binds to and deacetylates HIF-1120572 atlysine 674 This interaction blocks p300 recruitment to thepromoter of HIF-1120572 target genes and thereby represses HIF-1120572 transcriptional activity whereas Laemmle et al reportedthat SIRT1 increased HIF-1120572 protein levels [19]

In contrast Dioum et al [17] have reported that SIRT1does not target HIF-1120572 it rather deacetylates HIF-2120572 andtheir interaction promotes HIF-2120572 transcriptional activityIn addition because SIRT1 is a redox cellular sensor anddependent on metabolic status of the cell its regulation byhypoxia has been a point of interest In one report SIRT1is downregulated in hypoxic conditions due to decreasedNAD+ levels [18] while in another study it is upregulated ina HIF-dependent manner [39]

Thus the interaction between SIRT1 and HIF factors andthe resulting outcome of their interactions are still unclearIn this study we found that HIF-1120572 transcriptional activity isimpaired by SIRT1 inhibition under hypoxic conditions asreported in [19]

5 Conclusions

Our results suggest that SIRT1 exert a role in angiogenicproperties of hMSCs Thus our study might have importantimplications in the field of angiogenesis possibly leading tothe identification of chemical compounds that can positivelyregulate SIRT1 improving regenerative processes exerted byhMSCs

Conflict of Interests

The authors have stated that they have no conflict of interests

Acknowledgments

The authors are grateful to Dr Antonio Barbieri Dr GiosuersquoScognamiglio and Dr Giuseppina Liguori for microscopeassistance Special thanks are due to Dr Gabriella Malzoneand Dr Monica Cantile for important suggestions Theauthors are grateful to PON01 01227 (Botti Chiara) whichgave them the support needed to perform the research

References

[1] G Ren X Chen F Dong et al ldquoConcise review mesenchymalstem cells and translational medicine emerging issuesrdquo StemCells Translational Medicine vol 1 no 1 pp 51ndash58 2012

[2] C Stamm B Westphal H D Kleine et al ldquoAutologous bone-marrow stem-cell transplantation for myocardial regenerationrdquoThe Lancet vol 361 no 9351 pp 45ndash46 2003

[3] A Schiavetta CMaione C Botti et al ldquoA phase II trial of autol-ogous transplantation of bone marrow stem cells for criticallimb ischemia results of theNaples andPietra Ligure evaluationof stem cells studyrdquo Stem Cells Translational Medicine vol 1 no7 pp 572ndash578 2012

[4] T Kinnaird E Stabile M S Burnett et al ldquoMarrow-derivedstromal cells express genes encoding a broad spectrumof arteri-ogenic cytokines and promote in vitro and in vivo arteriogenesisthrough paracrine mechanismsrdquo Circulation Research vol 94no 5 pp 678ndash685 2004

[5] J Rehman D Traktuev J Li et al ldquoSecretion of angiogenicand antiapoptotic factors by human adipose stromal cellsrdquoCirculation vol 109 no 10 pp 1292ndash1298 2004

[6] H M Mi Y K Sun J K Yeon et al ldquoHuman adipose tissue-derived mesenchymal stem cells improve postnatal neovascu-larization in a mouse model of hindlimb ischemiardquo CellularPhysiology and Biochemistry vol 17 no 5-6 pp 279ndash290 2006

[7] R Estrada N Li H Sarojini J An M Lee and E WangldquoSecretome frommesenchymal stem cells induces angiogenesisvia Cyr61rdquo Journal of Cellular Physiology vol 219 no 3 pp 563ndash571 2009

[8] G Cobellis C Maione C Botti et al ldquoBeneficial effects ofVEGF secreted from stromal cells in supporting endothelial cellfunctions therapeutic implications for critical limb ischemiardquoCell Transplantation vol 19 no 11 pp 1425ndash1437 2010

[9] R H Lee J Y Oh H Choi and N Bazhanov ldquoTherapeuticfactors secreted bymesenchymal stromal cells and tissue repairrdquoJournal of Cellular Biochemistry vol 112 no 11 pp 3073ndash30782011

[10] P Simic K Zainabadi E Bell et al ldquoSIRT1 regulates differen-tiation of mesenchymal stem cells by deacetylating 120573-cateninrdquoEMBOMolecular Medicine vol 5 no 3 pp 430ndash440 2013

[11] A Z Herskovits and L Guarente ldquoSirtuin deacetylases inneurodegenerative diseases of agingrdquo Cell Research vol 23 no6 pp 746ndash758 2013

[12] MCHaigis andDA Sinclair ldquoMammalian sirtuins Biologicalinsights and disease relevancerdquo Annual Review of PathologyMechanisms of Disease vol 5 pp 253ndash295 2010

[13] T Finkel C Deng and RMostoslavsky ldquoRecent progress in thebiology and physiology of sirtuinsrdquo Nature vol 460 no 7255pp 587ndash591 2009

[14] L Guarente ldquoSirtuins aging and medicinerdquo The New EnglandJournal of Medicine vol 364 no 23 pp 2235ndash2244 2011

[15] V Carafa A Nebbioso and L Altucci ldquoSirtuins and disease theroad aheadrdquo Frontiers in Pharmacology vol 3 article 4 ArticleID Article 4 2012

[16] G Corbi A Bianco V Turchiarelli et al ldquoPotentialmechanismslinking atherosclerosis and increased cardiovascular risk inCOPD focus on sirtuinsrdquo International Journal of MolecularSciences vol 14 no 6 pp 12696ndash12713 2013

[17] E M Dioum R Chen M S Alexander et al ldquoRegulationof hypoxia-inducible factor 2alpha signaling by the stress-responsive deacetylase sirtuin 1rdquo Science vol 324 no 5932 pp1289ndash1293 2009

[18] J H Lim Y M Lee Y S Chun J Chen J Kim and JPark ldquoSirtuin 1 modulates cellular responses to hypoxia bydeacetylating hypoxia-inducible factor 1alphardquo Molecular Cellvol 38 no 6 pp 864ndash878 2010


[19] A Laemmle A Lechleiter V Roh et al ldquoInhibition of SIRT1impairs the accumulation and transcriptional activity of HIF-1120572 protein under hypoxic conditionsrdquo PLoS ONE vol 7 no 3Article ID e33433 2012

[20] C W Pugh and P J Ratcliffe ldquoRegulation of angiogenesis byhypoxia role of the HIF systemrdquo Nature Medicine vol 9 no 6pp 677ndash684 2003

[21] M Potente LGhaeni D Baldessari et al ldquoSIRT1 controls endo-thelial angiogenic functions during vascular growthrdquoGenes andDevelopment vol 21 no 20 pp 2644ndash2658 2007

[22] H Ota M Akishita M Eto K Iijima M Kaneki and YOuchi ldquoSirt1 modulates premature senescence-like phenotypein human endothelial cellsrdquo Journal of Molecular and CellularCardiology vol 43 no 5 pp 571ndash579 2007

[23] S Portmann R Fahrner A Lechleiter et al ldquoAntitumor effectof SIRT1 inhibition in human HCC tumor models in vitro andin vivordquoMolecular Cancer Therapeutics vol 12 no 4 pp 499ndash508 2013

[24] D Rotili D Tarantino A Nebbioso et al ldquoDiscovery ofsalermide-related sirtuin inhibitors binding mode studies andantiproliferative effects in cancer cells including cancer stemcellsrdquo Journal of Medicinal Chemistry vol 55 no 24 pp 10937ndash10947 2012

[25] I Gorenne S Kumar K Gray et al ldquoVascular smooth mus-cle cell sirtuin 1 protects against dna damage and inhibitsatherosclerosisrdquo Circulation vol 127 no 3 pp 386ndash396 2013

[26] M Potente ldquoAn energy-sensor network takes center stageduring endothelial agingrdquo Circulation Research vol 106 no 8pp 1316ndash1318 2010

[27] M Miceli G Franci C DellrsquoAversana et al ldquoMePR a novelhuman mesenchymal progenitor model with characteristics ofpluripotencyrdquo Stem Cells and Development vol 22 no 17 pp2368ndash2383 2013

[28] A G Bodnar M Ouellette M Frolkis et al ldquoExtension of life-span by introduction of telomerase into normal human cellsrdquoScience vol 279 no 5349 pp 349ndash352 1998

[29] T Okamoto T Aoyama T Nakayama et al ldquoClonal hetero-geneity in differentiation potential of immortalized humanmesenchymal stem cellsrdquo Biochemical and Biophysical ResearchCommunications vol 295 no 2 pp 354ndash361 2002

[30] S S Akimov A Ramezani T S Hawley and R G HawleyldquoBypass of senescence immortalization and transformation ofhuman hematopoietic progenitor cellsrdquo Stem Cells vol 23 no9 pp 1423ndash1433 2005

[31] M F Oellerich andM Potente ldquoFOXOs and sirtuins in vasculargrowth maintenance and agingrdquo Circulation Research vol 110no 9 pp 1238ndash1251 2012

[32] J Gracia-Sancho G Villarreal Y Zhang and G Garcıa-Cardena ldquoActivation of SIRT1 by resveratrol induces KLF2expression conferring an endothelial vasoprotective pheno-typerdquo Cardiovascular Research vol 85 no 3 pp 514ndash519 2010

[33] A Cardus A K Uryga G Walters and J D ErusalimskyldquoSIRT6 protects human endothelial cells from DNA dam-age telomere dysfunction and senescencerdquo CardiovascularResearch vol 97 no 3 pp 571ndash579 2013

[34] E Langley M Pearson M Faretta et al ldquoHuman SIR2 deacet-ylates p53 and antagonizes PMLp53-induced cellular senes-cencerdquo EMBO Journal vol 21 no 10 pp 2383ndash2396 2002

[35] H Ota E Tokunaga K Chang et al ldquoSirt1 inhibitor Sirtinolinduces senescence-like growth arrest with attenuated Ras-MAPK signaling in human cancer cellsrdquo Oncogene vol 25 no2 pp 176ndash185 2006

[36] S D Barros S Dehez E Arnaud et al ldquoAging-related decreaseof human ASC angiogenic potential is reversed by hypoxiapreconditioning through ROS productionrdquoMolecular Therapyvol 21 no 2 pp 399ndash408 2013

[37] N Engel andUMahlknecht ldquoAging and anti-aging unexpectedside effects of everyday medication through sirtuin 1 modula-tionrdquo International Journal of Molecular Medicine vol 21 no 2pp 223ndash232 2008

[38] M E Hubbi H Hu D M Gilkes and G L Semenza ldquoSirtuin-7 inhibits the activity of hypoxia-inducible factorsrdquo Journal ofBiological Chemistry vol 288 no 29 pp 20768ndash20775 2013

[39] R Chen M Xu R T Hogg et al ldquoThe acetylasedeacetylasecouple CREB-binding proteinsirtuin 1 controls hypoxia-inducible factor 2 signalingrdquo The Journal of Biological Chem-istry vol 287 no 36 pp 30800ndash30811 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


cytosol SIRT3 SIRT4 and SIRT5 are mitochondrial proteins[12] Sirtuins are generally known to regulate the acetylationlevels and the activity of histone and nonhistone regulatoryproteins

To date sirtuins have emerged as potential therapeutictargets for treatment of human pathologies such as cardio-vascular disease inflammation and cancer [15] SIRT1 is themost studied member of sirtuins It acts in various cellularprocesses and exerts its action activating and deactivatingfactors such as NF-120581B p53 p73 SOD and hypoxia-inducibletranscription factors (HIFs) [16ndash19]

Since SIRT1 targets several proteins in distinct signalingpathways modulation of SIRT1 activity alters the biologicalactivity of entire signaling networks modifying disease pro-gression such as pathological angiogenesis or atherosclerosis

An important component of pathological angiogenesis isrepresented by hypoxia that alters the cellular redox state andactivates SIRT1 In addition hypoxia works through multiplepathways to regulate angiogenesis for instance through themodulation of secreted angiogenic proteins such as vascularendothelial growth factor (VEGF) stimulated by increasedexpression of transcription factors such as HIF-1120572 [20]

Furthermore a ldquoprotectiverdquo role of SIRT1 in endothelialcells was described [21 22] A recent report has investigatedthe function of SIRT1 in regulating the differentiation ofmesenchymal stem cells by deacetylating 120573-catenin in mice[10] Several studies demonstrated that inhibition of SIRT1impairs cell growth in cancer cells [23 24] Gorenne et al[25] reported that SIRT1 expression was reduced in humanatherosclerotic plaques and in vascular smooth muscle cellsHowever to the best of our knowledge the effects of SIRT1modulation on angiogenesis in hMSCs have not been studiedyet

In this study we described the generation of an immor-talized human mesenchymal bone marrow-derived cell line(MeBM) and we investigated whether SIRT1 has an effect onangiogenic capability of hMSCs by inhibiting SIRT1 throughpharmacological and genetic approaches

Since recent studies have identified SIRT1 as a criticalmodulator of angiogenesis [18 26] we tested whether theinhibition of SIRT1 activity is associated with the reduction ofangiogenic ability of hMSCs and impaired hypoxic responsein these settings We found that the inhibition of SIRT1 activ-ity resulted in reduced capacity to proliferate to migrate andto form three-dimensional networks of vessel-like structuresIn addition SIRT1 inhibition reduced the hypoxia-inducedaccumulation of HIF-1120572 and its transcriptional activity inhMSCs

Our results suggested that SIRT1 is involved in angiogenicresponse of hMSCs in vitro and modulates the hypoxicresponse through inhibiting HIF-1120572 activity

Our findings may help to understand the role of SIRT1 inhMSCs in promoting angiogenesis and may contribute to thedevelopment of new strategies to improve the hMSCs-basedregenerative effects by modulating SIRT1 activity

2 Methods

21 Reagents Sirtinol was purchased from Selleck ChemicalsLLC (Houston TX USA) Culture medium and its supple-ments including antibiotics and fetal bovine serum (FBS)were purchased from Euroclone (Italy) Primary antibodiesagainst SIRT1 (Abcam Cambridge UK) HIF-1120572 (Santa CruzBiotechnology Santa Cruz CA USA) and tubulin (Sigma-Aldrich Milan Italy) were used Sirtinol was dissolved indimethyl sulfoxide (DMSO Sigma-Aldrich) to the appropri-ate concentrations according to reported procedures DMSOwas also present in the corresponding control

22 Cell Lines and Culture Medium Human mesenchymalstem cells (hMSCs) were obtained from bone marrow asdescribed by Cobellis et al [8] Cells were plated in RPMI1640 growth medium (Euroclone SPA Italy) containing 10heat-inactivated FBS 1 Pen-strep and 1 L-Glutamine

Cells were maintained as monolayers in a humidifiedatmosphere containing 5 CO

2at 37∘C and the culture

medium was replaced every two daysHypoxic culture conditionswere achieved in aBDGasPak

EZAnaerobeGasGenerating Pouch System (BDBiosciencesSan Diego) As certified by the manufacturer the AnaerobeGas Generating Pouch System produces an atmospherecontaining 10 carbon dioxide and 1 oxygen

Starvation conditions were obtained incubating cells inRPMI 1640 containing 02 FBS

23 Infection After plating bone marrow cells were grownto confluence and coinfected with HPV16 E6E7 and hTERTlentiviral vectors (infection number 1) After a week thecells were split and infected again only with hTERT (infec-tion number 2) and cultured until stabilization Sampleswere observed and photographed with DMI 6000 invertedmicroscope (Leica Microsystems) using Leica LAS ImageAnalysis software (Leica Microsystems) The protocol wasalso reported in Miceli et al [27]

24 hPV16 E6E7 and hTERT Lentiviral Production HIV-1based SIN lentiviral vectors were derived from SINF-MU3-W-S vector backbone hPV16 E6E7 was inserted upstreamof an encephalomyocarditis virus internal ribosome entrysite- (IRES-) yellow fluorescent protein (YFP) gene cassetteinto SINF-MU3-W-S to generate SINF-MU3-E6E7-IRES-YFPW-S SINF-MU3-hTERT-IRES-GFPW-S was generatedby inserting hTERT cDNAupstream of an IRES-green fluore-scent protein (GFP) gene cassette into SINF-MU3-W-S VSV-G-pseudotyped lentiviral vectors were generated in 150mmtissue culture dishes by transient cotransfectionwith (1) VSV-G-expressing construct pCMV-VSV-G (Invitrogen USA)(66 120583g) (2) packaging construct pCMVΔR82 (addgene)(48 120583g) and (3) lentiviral vector plasmids (pSin hTERT orPsin E6-E7) (66 120583g) into subconfluent HEK 293FT cells(Invitrogen) by calcium phosphate precipitation (ClontechCalphos Mammalian Transfection Kit) The supernatantcontaining the virus was produced in HEK-293FT collectedfiltered and used to infect bone marrow cells



2fully





2for 6 h





4Fe(CN)

6 5mM K

3Fe(CN)

6 150mMNaCl










3 Results






2or 1 O

















0

000005

00001

000015

00002

000025

00003m

RNA

expr

essio

n le

vels


lowast

NH

lowast

(a)

N H

SIRT1

Tubulin

(b)


(c)


(d)

0

10

20

30

40

50

60

70

80

90

sirtinolCTR

sirtinol

lowast

lowast

100120583M50120583M

SA-120573

-gal

pos

itive

cells

()

(e)


2(N) or 1 O








0

5

10

15

20

25

30

35

40

lowast

05

101520253035404550

lowast

CTR

RLU

s

RLU

s

0

10

20

60

lowast

RLU

s

24h



48h

72h

30

40

50

times104

times104 times104

(a)

0

1

2

3

4

5

6

7

8

9

RLU

s

sh-Sirt1

lowast

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

CTR

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

48h24h

72htimes104

times104 times104

(b)

Figure 2 Continued


01

655

150

193

79525160216

10758120119

50

684

145

167

0275598146

48

566

211

220

CTR

48h

600

500

400

300

200

100

0

400

320

240

160

80

0

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

Num

ber

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

400

300

200

100

0

Num

ber

24h

72h

100120583M sirtinol

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

(c)



0

5

10

15

20

25

0

5

10

15

20

25

30

35

40

45

Mig

rate

d ce

lls (

)

CTR0

5

10

15

20

25

30

35

CTR

CTR

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

24h 48h

72h


100120583M sirtinol

lowastlowast

lowast

(a)

0

2

4

6

8

10

12

CTR sh-Sirt10

2

4

6

8

10

12

14

CTR sh-Sirt1

0

2

4

6

8

10

12

14

16

CTR sh-Sirt1

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

lowast

lowast

lowast

24h 48h

72h

(b)

Figure 3 Continued




(c)

0

5

10

15

20

25

Num

ber o

f bra

nch

poin

ts (



(d)







2or 1 O



4 Discussion





SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2fully





2for 6 h





4Fe(CN)

6 5mM K

3Fe(CN)

6 150mMNaCl










3 Results






2or 1 O

















0

000005

00001

000015

00002

000025

00003m

RNA

expr

essio

n le

vels


lowast

NH

lowast

(a)

N H

SIRT1

Tubulin

(b)


(c)


(d)

0

10

20

30

40

50

60

70

80

90

sirtinolCTR

sirtinol

lowast

lowast

100120583M50120583M

SA-120573

-gal

pos

itive

cells

()

(e)


2(N) or 1 O








0

5

10

15

20

25

30

35

40

lowast

05

101520253035404550

lowast

CTR

RLU

s

RLU

s

0

10

20

60

lowast

RLU

s

24h



48h

72h

30

40

50

times104

times104 times104

(a)

0

1

2

3

4

5

6

7

8

9

RLU

s

sh-Sirt1

lowast

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

CTR

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

48h24h

72htimes104

times104 times104

(b)

Figure 2 Continued


01

655

150

193

79525160216

10758120119

50

684

145

167

0275598146

48

566

211

220

CTR

48h

600

500

400

300

200

100

0

400

320

240

160

80

0

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

Num

ber

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

400

300

200

100

0

Num

ber

24h

72h

100120583M sirtinol

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

(c)



0

5

10

15

20

25

0

5

10

15

20

25

30

35

40

45

Mig

rate

d ce

lls (

)

CTR0

5

10

15

20

25

30

35

CTR

CTR

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

24h 48h

72h


100120583M sirtinol

lowastlowast

lowast

(a)

0

2

4

6

8

10

12

CTR sh-Sirt10

2

4

6

8

10

12

14

CTR sh-Sirt1

0

2

4

6

8

10

12

14

16

CTR sh-Sirt1

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

lowast

lowast

lowast

24h 48h

72h

(b)

Figure 3 Continued




(c)

0

5

10

15

20

25

Num

ber o

f bra

nch

poin

ts (



(d)







2or 1 O



4 Discussion





SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2or 1 O

















0

000005

00001

000015

00002

000025

00003m

RNA

expr

essio

n le

vels


lowast

NH

lowast

(a)

N H

SIRT1

Tubulin

(b)


(c)


(d)

0

10

20

30

40

50

60

70

80

90

sirtinolCTR

sirtinol

lowast

lowast

100120583M50120583M

SA-120573

-gal

pos

itive

cells

()

(e)


2(N) or 1 O








0

5

10

15

20

25

30

35

40

lowast

05

101520253035404550

lowast

CTR

RLU

s

RLU

s

0

10

20

60

lowast

RLU

s

24h



48h

72h

30

40

50

times104

times104 times104

(a)

0

1

2

3

4

5

6

7

8

9

RLU

s

sh-Sirt1

lowast

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

CTR

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

48h24h

72htimes104

times104 times104

(b)

Figure 2 Continued


01

655

150

193

79525160216

10758120119

50

684

145

167

0275598146

48

566

211

220

CTR

48h

600

500

400

300

200

100

0

400

320

240

160

80

0

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

Num

ber

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

400

300

200

100

0

Num

ber

24h

72h

100120583M sirtinol

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

(c)



0

5

10

15

20

25

0

5

10

15

20

25

30

35

40

45

Mig

rate

d ce

lls (

)

CTR0

5

10

15

20

25

30

35

CTR

CTR

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

24h 48h

72h


100120583M sirtinol

lowastlowast

lowast

(a)

0

2

4

6

8

10

12

CTR sh-Sirt10

2

4

6

8

10

12

14

CTR sh-Sirt1

0

2

4

6

8

10

12

14

16

CTR sh-Sirt1

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

lowast

lowast

lowast

24h 48h

72h

(b)

Figure 3 Continued




(c)

0

5

10

15

20

25

Num

ber o

f bra

nch

poin

ts (



(d)







2or 1 O



4 Discussion





SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

000005

00001

000015

00002

000025

00003m

RNA

expr

essio

n le

vels


lowast

NH

lowast

(a)

N H

SIRT1

Tubulin

(b)


(c)


(d)

0

10

20

30

40

50

60

70

80

90

sirtinolCTR

sirtinol

lowast

lowast

100120583M50120583M

SA-120573

-gal

pos

itive

cells

()

(e)


2(N) or 1 O








0

5

10

15

20

25

30

35

40

lowast

05

101520253035404550

lowast

CTR

RLU

s

RLU

s

0

10

20

60

lowast

RLU

s

24h



48h

72h

30

40

50

times104

times104 times104

(a)

0

1

2

3

4

5

6

7

8

9

RLU

s

sh-Sirt1

lowast

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

CTR

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

48h24h

72htimes104

times104 times104

(b)

Figure 2 Continued


01

655

150

193

79525160216

10758120119

50

684

145

167

0275598146

48

566

211

220

CTR

48h

600

500

400

300

200

100

0

400

320

240

160

80

0

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

Num

ber

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

400

300

200

100

0

Num

ber

24h

72h

100120583M sirtinol

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

(c)



0

5

10

15

20

25

0

5

10

15

20

25

30

35

40

45

Mig

rate

d ce

lls (

)

CTR0

5

10

15

20

25

30

35

CTR

CTR

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

24h 48h

72h


100120583M sirtinol

lowastlowast

lowast

(a)

0

2

4

6

8

10

12

CTR sh-Sirt10

2

4

6

8

10

12

14

CTR sh-Sirt1

0

2

4

6

8

10

12

14

16

CTR sh-Sirt1

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

lowast

lowast

lowast

24h 48h

72h

(b)

Figure 3 Continued




(c)

0

5

10

15

20

25

Num

ber o

f bra

nch

poin

ts (



(d)







2or 1 O



4 Discussion





SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

30

35

40

lowast

05

101520253035404550

lowast

CTR

RLU

s

RLU

s

0

10

20

60

lowast

RLU

s

24h



48h

72h

30

40

50

times104

times104 times104

(a)

0

1

2

3

4

5

6

7

8

9

RLU

s

sh-Sirt1

lowast

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

CTR

0

2

4

6

8

10

12

14

16

18

lowast

sh-Sirt1CTR

RLU

s

48h24h

72htimes104

times104 times104

(b)

Figure 2 Continued


01

655

150

193

79525160216

10758120119

50

684

145

167

0275598146

48

566

211

220

CTR

48h

600

500

400

300

200

100

0

400

320

240

160

80

0

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

Num

ber

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

400

300

200

100

0

Num

ber

24h

72h

100120583M sirtinol

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

(c)



0

5

10

15

20

25

0

5

10

15

20

25

30

35

40

45

Mig

rate

d ce

lls (

)

CTR0

5

10

15

20

25

30

35

CTR

CTR

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

24h 48h

72h


100120583M sirtinol

lowastlowast

lowast

(a)

0

2

4

6

8

10

12

CTR sh-Sirt10

2

4

6

8

10

12

14

CTR sh-Sirt1

0

2

4

6

8

10

12

14

16

CTR sh-Sirt1

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

lowast

lowast

lowast

24h 48h

72h

(b)

Figure 3 Continued




(c)

0

5

10

15

20

25

Num

ber o

f bra

nch

poin

ts (



(d)







2or 1 O



4 Discussion





SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















01

655

150

193

79525160216

10758120119

50

684

145

167

0275598146

48

566

211

220

CTR

48h

600

500

400

300

200

100

0

400

320

240

160

80

0

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

0 50 100 150 200 250

Channels (FL2-A)

Num

ber

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

240

180

120

60

0

Num

ber

400

300

200

100

0

Num

ber

24h

72h

100120583M sirtinol

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

ltG1()G1 ()G2 ()S ()

(c)



0

5

10

15

20

25

0

5

10

15

20

25

30

35

40

45

Mig

rate

d ce

lls (

)

CTR0

5

10

15

20

25

30

35

CTR

CTR

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

24h 48h

72h


100120583M sirtinol

lowastlowast

lowast

(a)

0

2

4

6

8

10

12

CTR sh-Sirt10

2

4

6

8

10

12

14

CTR sh-Sirt1

0

2

4

6

8

10

12

14

16

CTR sh-Sirt1

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

lowast

lowast

lowast

24h 48h

72h

(b)

Figure 3 Continued




(c)

0

5

10

15

20

25

Num

ber o

f bra

nch

poin

ts (



(d)







2or 1 O



4 Discussion





SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

0

5

10

15

20

25

30

35

40

45

Mig

rate

d ce

lls (

)

CTR0

5

10

15

20

25

30

35

CTR

CTR

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

24h 48h

72h


100120583M sirtinol

lowastlowast

lowast

(a)

0

2

4

6

8

10

12

CTR sh-Sirt10

2

4

6

8

10

12

14

CTR sh-Sirt1

0

2

4

6

8

10

12

14

16

CTR sh-Sirt1

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

Mig

rate

d ce

lls (

)

lowast

lowast

lowast

24h 48h

72h

(b)

Figure 3 Continued




(c)

0

5

10

15

20

25

Num

ber o

f bra

nch

poin

ts (



(d)







2or 1 O



4 Discussion





SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(c)

0

5

10

15

20

25

Num

ber o

f bra

nch

poin

ts (



(d)







2or 1 O



4 Discussion





SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SIRT1

Tubulin

HIF1120572

CTR 100120583M

1 O2

(a)

0

00002

00004

00006

00008

0001

00012

00014

00016

00018

SIRT1 VEGF GLUT1

mRN

A ex

pres

sion

leve

ls

NHH + sirtinol

lowast lowast

lowast

(b)
























5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusions




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article SIRT1 Inhibition Affects Angiogenic...

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