Research Article Diabetes-Induced Oxidative Stress in...

Research ArticleDiabetes-Induced Oxidative Stress in EndothelialProgenitor Cells May Be Sustained by a Positive FeedbackLoop Involving High Mobility Group Box-1

Han Wu Ran Li Zhong-Hai Wei Xin-Lin Zhang Jian-Zhou ChenQing Dai Jun Xie and Biao Xu

Department of Cardiology Drum Tower Hospital Nanjing University Medical School Nanjing 210008 China

Correspondence should be addressed to Jun Xie hsp70xjgmailcom and Biao Xu submittingpaper163com

Received 20 July 2015 Revised 24 September 2015 Accepted 28 September 2015

Academic Editor Umesh Yadav

Copyright copy 2016 Han Wu et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Oxidative stress is considered to be a critical factor in diabetes-induced endothelial progenitor cell (EPC) dysfunction although theunderlying mechanisms are not fully understood In this study we investigated the role of high mobility group box-1 (HMGB-1)in diabetes-induced oxidative stress HMGB-1 was upregulated in both serum and bone marrow-derived monocytes from diabeticmice compared with control mice In vitro advanced glycation end productions (AGEs) induced expression of HMGB-1 in EPCsand in cell culture supernatants in a dose-dependent manner However inhibition of oxidative stress with N-acetylcysteine (NAC)partially inhibited the induction of HMGB-1 induced by AGEs Furthermore p66shc expression in EPCs induced by AGEs wasabrogated by incubation with glycyrrhizin (Gly) while increased superoxide dismutase (SOD) activity in cell culture supernatantswas observed in the Gly treated groupThus HMGB-1 may play an important role in diabetes-induced oxidative stress in EPCs viaa positive feedback loop involving the AGEreactive oxygen speciesHMGB-1 pathway

1 Introduction

Diabetes mellitus (DM) has been widely recognized as animportant modern-day disease and cardiovascular compli-cations are the leading causes of morbidity and mortality inDMpatients Impaired angiogenesis is thought to be a criticalevent contributing to the development of cardiovascularcomplications associated with diabetes [1] Notably there isagreement in the literature that endothelial progenitor cells(EPCs) the precursors of endothelial cells may contributeto angiogenesis and endothelial repair [2 3] However thenumber of EPCs is reduced and the function is impairedin patients with diabetes with EPCs found to be defectivein vascular repair thus contributing to the progression ofcardiovascular disease in this patients [4ndash6]

Oxidative stress is a major cause of various pathologicalprocesses in DM [7] Early reports demonstrated that thenumber of EPCs was negatively correlated with oxidativestress which resulted in diabetes-related EPC dysfunction[8] Thus oxidative stress may represent an important

therapeutic target in the prevention of impaired vascularhomeostasis in DM Although discoveries made in the lastdecade have made it clear that oxidative stress is central toimpaired angiogenesis the endogenous mechanisms remainpoorly understood

Increasing evidence demonstrates that EPCs can bedivided into two types early and late EPCs Early EPCswhichare generated by the culture of peripheral bloodmononuclearcells in medium for approximately 4 days exhibit higherlevels of cytokine release and lower vessel growth comparedwith late EPCs Interestingly late EPCs obtained by long-termculture of early EPCs show greater vasculogenic potential andare regarded as ldquotrue EPCsrdquo [9]Thus late EPCs were used toevaluate the effects of oxidative stress in this investigation

It has been established that hyperglycemia promotesreactions between plasma proteins and glucose through anonenzymatic process leading to the formation of advancedglycation endproductions (AGEs) AGEs are considered to beimportant mediators of diabetes and diabetic complicationsIn diabetes AGEs accumulate in tissues at an accelerated

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 1943918 9 pageshttpdxdoiorg10115520161943918

2 Oxidative Medicine and Cellular Longevity

rate and then contribute at least in part to the initiationand development of diabetic cardiovascular complications[10ndash12] In the past decade accumulating evidence hasshown that AGEs promote oxidative stress in EPCs and thenmediate EPC dysfunction in processes such as migrationtube formation and apoptosis [13 14]

High mobility group box-1 (HMGB-1) a nonchromo-somal nuclear protein is ubiquitously expressed in variouscells including monocytes cardiomyocytes and endothelialcells Once released into the serum in response to stressessuch as high glucose conditions HMGB-1 functions as aproinflammatory cytokine Recent studies have indicated thatoxidative stress promotes HMGB-1 release in various cells[15] resulting in the induction of reactive oxygen species(ROS) production in cardiomyocytes [16] However the roleof HMGB-1 in diabetes-induced oxidative stress in late EPCshas received little attention Thus we hypothesized thatdiabetes induces the release of HMGB-1 which subsequentlyenhances oxidative stress in late EPCs In the present studyHMGB-1 expression in serum and monocytes of diabeticmice was analyzed and the role of HMGB-1 in AGE-inducedoxidative stress in late EPCs was investigated in vitro

2 Methods

21 Induction and Assessment of Diabetes The experimentaland feeding protocols were approved and conducted in accor-dance with the laws and regulations controlling experimentson live animals in China and the Asian Convention forthe Protection of Vertebrate Animals used for Experimentaland Other Scientific Purposes Male C57BL6 mice werepurchased from the Model Animal Research Center of Nan-jing University (China) Before injection of streptozotocin(STZ) the mice were weighed and the blood glucose wasdetected in a sample obtained from the tail vein using bloodglucose monitoring system Diabetes was induced in mice byconsecutive intraperitoneal injections of STZ (40mgkgdaySigma-Aldrich USA) for 5 days After 3 days of STZ injectionblood glucose was determined as described previously Micewith blood glucose levels gt139mmoLl in three consecutivemeasurements were considered to be diabetic Mice treatedwith citrate buffer were used as nondiabetic controls Sixteenmice were divided into the following two groups normalnondiabetic group (N group 119899 = 8) and diabetic group (DMgroup 119899 = 8) Nonfasting blood glucose and weight weremeasured every month until the end of the experiment

22Measurement of SerumHMGB-1 Levels At the end of thisinvestigation the mice were anesthetized and sacrificed bydecapitation and blood was collected for serum separationSerum HMGB-1 concentrations were determined using amouse HMGB-1 ELISA kit (USCN Life Science China)according to the manufacturerrsquos instructions

23 Isolation of Monocytes from Mouse Bone Marrow Afterthe mice were sacrificed the femurs and tibias were immedi-ately excised and flushed with cold PBS After centrifugationat 1400 rpm for 5min the collected cells were filtered and

resuspended in red blood cell lysing buffer After 5min thecells were centrifuged at 1500 rpm for 5min After threewashing steps the monocytes were used in Western blotanalyses

24 EPC Isolation and Characterization The protocol wasapproved by the Ethical Committee of Institutional EthicsCommittee of Nanjing University Medical School (China)EPCs were isolated from the peripheral blood of healthyvolunteers as previously described [14 17] Briefly peripheralblood mononuclear cells (PBMCs) were collected by Ficoll-Paque PLUS (GE Healthcare Life Sciences USA) densitygradient centrifugation of peripheral blood PBMCs wereresuspended in endothelial cell growth medium-2 (EGM-2) (Lonza Switzerland) composed of endothelial cell basalmedium-2 (EBM-2) 5 fetal bovine serum (FBS) andgrowth factors containing vascular endothelial growth factor(VEGF) basic fibroblast growth factor (bFGF) epidermalgrowth factor (EGF) insulin-like factor-1 (IGF-1) hydrocor-tisone heparin and ascorbic acid After 4 days in culturemedium and nonadherent cells were removed the mediumwas replaced every 3 days After approximately 3 weeks theldquocobblestonerdquomorphology observed bymicroscope indicatedthat the cells were late EPCs These cells (passage lt 5) wereused in subsequent studies

To confirm the late EPC phenotype the cells were incu-bated with 5 120583gmL 11-dioctadecyl-3333-tetramethylin-docarbocyanine-labeled low density lipoprotein (Dil-acLDLMolecular Probes) for 2 h at 37∘C and then fixed with4 paraformaldehyde for 30min After being washed threetimes the cells were incubated with 10 120583gmL fluorescein-isothiocyanate-conjugated lectin (FITC-lectin Sigma USA)for 1 h at room temperature in the dark Additionally late EPCsurface markers were detected by immunocytochemistryusing FITC-CD34 (BD USA) allophycocyanin-KDR (APC-KDR RampD USA) and rabbit anti-CXCR4 (Abcam UK)Then goat anti-rabbit Alexa Fluor 488 was used to detect theexpression of CXCR4 Nuclei were stained with DAPI Thecells were observed by fluorescence microscopy (OlympusJapan)

25Western Blot Analysis After beingwashedwith cold PBSthe cells were resuspended in cell lysis buffer containing acocktail of protease inhibitors (1 100 Sigma USA) Aftercentrifugation at 15000 rpm for 10min the supernatant wascollected and stored at minus80∘C for later use The protein con-centration of the cell lysate was detected using a BCA proteinassay kit (Pierce USA) Total cell proteins or cell supernatantswere mixed with loading buffer and were heated in boilingwater for 10min The proteins were then separated by SDS-PAGE electrotransferred and blotted onto polyvinylidenedifluoride membranes The membranes were blocked with10 dried nonfat milk in 01 PBS-Tween-20 (PBST) for 2 hat room temperature and hybridized overnight at 4∘C withanti-HMGB-1 (1 1000 Bioworld Technology USA) anti-p66shc (1 1000 Santa Cruz USA) and anti-sirt1 (1 500Bioworld Technology USA) Membranes were then washedwith 01 PBST and incubated with appropriate secondary

Oxidative Medicine and Cellular Longevity 3

antibodies The reactions were developed using enhancedchemiluminescence reagents and images were obtained byexposure to film The bands were analyzed using BioRadQuantity One imaging software

26 Evaluation of Intracellular ROS Fluorescent probe 21015840-71015840-dichlorofluorescin diacetate (DCFH-DA Sigma USA) wasused to measure intracellular ROS generation in late EPCsplated in 6-well plates After different treatments themediumwas removed and the cells were washed with PBS The cellswere then incubated with 5 120583molL DCFH-DA probe inserum-free medium at 37∘C for 30min Intracellular ROSproduction was detected using a fluorescence microscope(Olympus Japan)

27 SuperoxideDismutase ActivityDetermination Thesuper-oxide dismutase (SOD) activity in cell supernatants wasevaluated using SOD assay kit (Jiancheng BioengineeringResearch Institute China) according to the manufacturerrsquosinstructions and was expressed as a percentage comparedwith the control group

28 Statistical Analysis Data were expressed as the meansplusmn standard deviation (SD) and were analyzed with one-wayANOVA using SPSS 200 software (SPSS lnc USA) 119875 lt 005was considered to indicate statistical significance

3 Results

31 Diabetes Induced IncreasedHMGB-1 in Serum and in BoneMarrow-DerivedMonocytes fromDiabeticMice As shown inFigure 1(a) diabetic mice exhibited increased blood glucosecompared with normal mice this high level was stablymaintained until the mice were sacrificed Furthermorediabetes caused deleterious inhibition of normal weight gainduring the present study (Figure 1(b)) Figure 1(c) showed theserum HMGB-1 levels in normal and diabetic mice STZ-induced diabetic mice showed significantly higher serumHMGB-1 levels than those of normal mice As monocytes arethe main source of serum HMGB-1 HMGB-1 expression inmonocytes from normal and diabetic mice was detected byWestern blot analysis showing that HMGB-1 protein levelswere significantly higher in monocytes from diabetic micethan those from nondiabetic mice (Figure 1(d))

32 AGEs Induced HMGB-1 Upregulation in Late EPCs andin Cell Culture Supernatants More than 90 of the attachedcells that were double positive for Dil-acLDL uptake andlectin staining were characterized as late EPCs Immunophe-notyping revealed that late EPCs expressed CD34 andCXCR4 the surface antigens of progenitor cells as well asKDR which is a characteristic surface marker of endothelialcell (Figure 2(a)) AGEs were used to establish an in vitrodiabetesmodel to investigate the role of HMGB-1 in diabetes-induced oxidative stress HMGB-1 levels were measured incell culture supernatants and in late EPCs exposed to AGEsIn order to screen the optimal stimulation concentration ofAGEs late EPCs were incubated with 50ndash400120583gmL AGEs

for 24 h using 400120583gmL BSA as a control As shown inFigure 2(b) AGEs induced the upregulation of HMGB-1 inlate EPCs in a dose-dependentmanner and the effect reachedthe level of statistical significance at 100 120583gmL In additionHMGB-1 in cell supernatants was significantly increased byexposure to AGEs at 100 120583gmL or 200120583gmL (Figure 2(c))Thus in subsequent experiments late EPCs were stimulatedwith 100 120583gmL AGEs for 24 h

33 Oxidative Stress Was Involved in AGE-Induced IncreasedHMGB-1 Expression To investigate the mechanism of AGE-induced oxidative stress in late EPCs we determinedthe expression of sirt1 and p66shc in late EPCs Ourresults showed that AGE-stimulated EPCs displayed sig-nificantly decreased sirt1 expression and this effect waspartly reversed by pretreatment with resveratrol an activatorof sirt1 (Figure 3(a)) p66shc a critical protein regulatingcellular oxidative stress responses is considered to be thedownstream of sirt1 Increased p66shc expression in AGE-stimulated EPCs was abrogated by resveratrol (Figure 3(b))In addition treatment with Ex527 a sirt1 antagonist reversedthe effects of resveratrol (Figure 3(b)) suggesting that thesirt1p66shc pathway modulated oxidative stress in late EPCsin response to AGEs

We next investigated whether AGEs induced HMGB-1expression in late EPCs via ROS production by using NACto inhibit oxidative stress induced in late EPCs by AGEs TheROS generation in late EPCs demonstrated by DCFH-DAstaining was significantly increased by AGE exposure thiseffect was abrogated by N-acetylcysteine (NAC) treatment(Figure 3(c)) NAC abolished the stimulatory effect of AGEsonHMGB-1 expression in late EPCs (Figure 3(d)) suggestingthat AGEs induce HMGB-1 expression in late EPCs viaoxidative stress

34 Inhibition ofHMGB-1AttenuatedOxidative Stress Inducedby AGEs in Late EPCs Glycyrrhizin was used to block thestimulation ofHMGB-1 to determine its role inAGE-inducedoxidative stress detected by intracellular p66shc expressionand superoxide dismutase (SOD) activity in cell culturesupernatants Treatment of late EPCs with glycyrrhizinabrogated AGE-induced upregulation of p66shc expres-sion (Figure 4(a)) Furthermore the decreased SOD activityinduced by AGEs was reversed by the glycyrrhizin admin-istration (Figure 4(b)) Similar to p66shc AGE-induced dys-regulation of sirt1 expressionwas suppressed by the inhibitionof HMGB-1 (Figure 4(c))

4 Discussion

In the present study we demonstrate for the first timethat AGEs induce HMGB-1 expression in late EPCs viasirt1p66shc-mediated oxidative stress and that the inhibitionof HMGB-1 attenuates sirt1p66shc pathway activity andoxidative stress in late EPCs exposed to AGEs Thus weconclude that HMGB-1 may amplify sirt1p66shc pathwaysignaling and oxidative stress reactions in late EPCs inducedby AGEs (Figure 5)


Bloo

d gl

ucos

e (m

mol

L)

0 1 2 3Month

6

7

8

9

26

28

30

32

NDM

lowastlowast

lowastlowast lowastlowast

(a)

0 1 2 3Month

Body

wei

ght (

g)

20

25

30

NDM

lowastlowast

lowastlowast

lowast

(b)

N DM

Seru

m H

MG

B-1

(pg

mL)

lowast

0

200

400

600

(c)N DM

120573-actin

HMGB-1H

MG

B-1120573

-act

in

lowast

00

05

10

15

20

(d)

Figure 1 Blood glucose body weight and HMGB-1 expression in normal and diabetic mice (a b) Blood glucose and body weight in normaland diabetic mice (c) Serum HMGB-1 levels in normal and diabetic mice (d) Expression of HMGB-1 in bone marrow-derived monocytesin normal and diabetic mice lowast119875 lt 005 and lowastlowast119875 lt 001 DM group versus N group (119899 = 8 per group) N normal mice DM diabetic mice

HMGB-1 is a nuclear DNA-binding protein that regulatesgene transcription and maintains the nucleosome structureUnder stress conditions HMGB-1 is released from the celland functions as a multifunctional cytokine that contributesto various pathophysiological processes

Accumulating clinical and experimental evidencedemonstrates the presence of elevated serum HMGB-1in diabetic patients and increased HMGB-1 expressionin diabetic animals [18ndash20] Furthermore a recent studyshowed that plasma HMGB-1 levels were increased inChinese subjects with pure type 2 DM [21] In accordance

with previous studies the current investigation demonstratedthat serum HMGB-1 levels were significantly higher in STZ-induced diabetic mice compared with their nondiabeticcounterparts It is worth mentioning that increased HMGB-1expression was also observed in bone marrow-derivedmonocytes from diabetic mice It can be speculated thatEPCs from diabetic mice also exhibited higher HMGB-1expression because the EPCs were predominantly mobilizedfrom bone marrow Although we did not detect expressionof HMGB-1 in EPCs from diabetic mice directly our in vitrostudies demonstrated that HMGB-1 expression increased


DAPI Dil-acLDL FITC-lectin Merge

BF CD34 KDR CXCR4

(a)

50 100 200 400

120573-actin

HMGB-1

HM

GB-

1120573

-act

in

00

05

10

15

20

25

BSA (400120583gmL)AGEs (120583gmL)

minusminusminusminusminus

minus minus

+

lowastlowast

lowastlowastlowastlowast

(b)

Cell supernatant

HMGB-1

HM

GB-

1120573

-act

in

0

1

2

3

4

50 100 200 400BSA (400120583gmL)

AGEs (120583gmL)minusminusminusminusminus

minus minus

+


(c)

Figure 2 Expression of HMGB-1 in late EPCs and cell supernatants induced by AGEs at different concentrations (a) Late EPCs were shownto endocytose Dil-acLDL and bind to lectin Late EPCs showed a typical ldquocobblestonerdquo morphology in phase-contrast inverted microscopyafter approximately 3 weeks of culture andwere positive for expression of CD34 KDR andCXCR4 (b) Effect of AGEs onHMGB-1 expressionin late EPCs (c) HMGB-1 protein expression in cell culture supernatants of late EPCs exposed to AGEs Scale bar = 20120583m lowastlowast119875 lt 001 versuscontrol group 119899 = 3 BF bright field BSA bovine serum albumin

significantly and dose dependently in late EPCs followingthe exposure to AGEs and was accompanied by an increasein the levels detected in cell culture supernatants This isin accordance with previous reports that hyperglycemiainduced HMGB-1 expression in endothelial cells vascularsmooth muscle cells and cardiomyocytes [22ndash24] Inaddition the underlying mechanism by which diabetesinduces HMGB-1 was investigated in this study

Oxidative stress has been reported to be involved in activeHMGB-1 secretion [15 25] and HMGB-1 in animal modelsis inhibited by antioxidants such as resveratrol [19 26] Asoxidative stress is a central step leading to EPC dysfunction indiabetes and thus contributes to cardiovascular impairment[1 27] we hypothesize that diabetes induces the upregulationand release of HMGB-1 in late EPCs via oxidative stressAs shown in Figure 3 production of both HMGB-1 and


sirt1

120573-actin

sirt1

120573-a

ctin

00

05

10

15

lowast

lowast

lowast

lowastlowast

AGEs (100120583gmL)minusminus minus

+ + + +

Res (120583M) 10 20 40

(a)p6

6shc

120573-a

ctin

p66shc

120573-actin

00

05

10

15

lowastlowast

lowast

20

NS


minus minus minus

minus

+ + +

+ +

+

+ +

Res (10 120583M)Ex527 (10120583M)

(b)

DCFH-DA

Control AGEs AGEs + NAC

(c)

HMGB-1

120573-actin

HM

GB-

1120573

-act

in

00

05

10

15

20

25

NAC (10mmolL) minus minus +

AGEs (100120583gmL) minus + +


(d)

Figure 3 Inhibition of oxidative stress by NAC attenuated HMGB-1 expression in late EPCs (a) AGE-induced downregulation of sirt1 waspartly normalized by resveratrol (b) Inhibition of sirt1 by Ex527 decreased p66shc expression in AGE-stimulated late EPCs (c) DCFH-DAstaining showing that pretreatment with NAC inhibited ROS production in late EPCs (d) NAC suppressed HMGB-1 expression in AGE-stimulated late EPCs lowast119875 lt 005 between the two groups and lowastlowast119875 lt 001 between the two groups NS no difference between the two groups119899 = 3 Scale bar = 25 120583m

ROS induced by AGEs in late EPCs was partially abolishedby treatment with NAC suggesting that oxidative stressis involved in AGE-induced HMGB-1 expression and thatHMGB-1 production occurs downstream of ROS productionin late EPCs stimulated by AGEs

RAGE belongs to an immunoglobulin superfamily of cellsurface molecules with the capacity to bind to a number ofligands such as AGEs HMGB-1 amyloid fibrils and S-100proteins and is involved in a variety of cellular functions [28]It is generally accepted that RAGE activation contributes to


p66s

hc120573

-act

inp66shc

120573-actin

Gly (120583molL) minus minus 50 100 200

AGEs (100120583gmL) minus + + + +

00

05

10

15

20

lowast

(a)

SOD

activ

ity (

)

Gly (120583molL)AGEs (100120583gmL)

minus

minus

minus

+

50

+

100

+

200

+

lowast

0

50

100

150

(b)

sirt1

120573-actin

sirt1

120573-a

ctin

Gly (100120583molL) minus minus +AGEs (100120583gmL) minus + +

00

05

10

15

lowastlowast

(c)

Figure 4 Glycyrrhizin normalized the dysregulated p66shc expression in AGEs treated late EPCs and the dysregulated SOD activity in cellculture supernatants (a) The increased p66shc expression induced by AGEs was abrogated by glycyrrhizin treatment (b) Treatment of lateEPCs with glycyrrhizin reversed the decreased SOD activity induced by AGEs (c) Glycyrrhizin attenuated the AGE-induced decrease in sirt1expression in late EPCs lowast119875 lt 005 versus control group lowastlowast119875 lt 001 versus control group 119875 lt 005 versus AGEs group and

119875 lt 001

versus AGE group 119899 = 3 Gly glycyrrhizin

oxidative stress in diabetes [13 29] HMGB-1 is a well-knownnon-AGE ligand for RAGE and can induce endothelialdysfunction [30] therefore HMGB-1 may cause increasedROS generation in late EPCs

Oxidative stress is defined as an imbalance between ROSgeneration and antioxidant defenses The adaptor proteinp66shc promotes ROS production [31 32] while SOD func-tions as an intracellular enzymatic antioxidant by scavenging

excess oxygen-free radicals Thus p66shc expression in lateEPCs and SOD activity in cell culture supernatants weredetermined to evaluate the effects of glycyrrhizin on oxidativestress induced in late EPCs by AGEs In accordance with thefindings of others [33 34] the present study showed dys-regulation of p66shc expression and SOD activity in AGEs-stimulated late EPCs Furthermore the changes in p66shcand SOD activity were partly normalized by inhibiting


RAGE

AGEHMGB-1

HMGB-1

Nucleus

Cytoplasm

Extracellular

p66shc uarr

sirt1 darr

ROS uarr

Figure 5 Hypothetical diagram of HMGB-1-mediated enhance-ment of oxidative stress induced in late EPCs by AGEs AGEsinduced ROS production via the sirt1p66shc pathway and then pro-moted HMGB-1 release from the late EPCs Extracellular HMGB-1 augmented ROS production via binding to RAGE and signalingthrough the sirt1p66shc pathway

HMGB-1 with glycyrrhizin suggesting that HMGB-1 medi-ates oxidative stress induced in late EPCs by AGEs Inaccordance with this speculation Feng demonstrated thatHMGB-1 mediated endothelial activation by oxidative stressresponses via the RAGE pathway [30] Furthermore Zhangrecently showed that HMGB-1 induced a marked increase inintracellular ROS in cardiomyocytes [16] All these observa-tions indicate that HMGB-1 stimulates oxidative stress in avariety of cell types

Sirt1 a mammalian homologue of sir2 was identified asa key mediator of various diseases Accumulating evidencesuggests that sirt1 promotes cellular resistance to oxidativestress associated with diabetes In the current investigationwe demonstrated that AGE treatment significantly decreasedsirt1 expression in late EPCs while resveratrol an activatorof sirt1 normalized the downregulated sirt1 expressionFurthermore resveratrol treatment inhibited AGE-inducedp66shc expression in late EPCs although the effect waslost when AGE-stimulated late EPCs were pretreated withthe sirt1 antagonist Ex527 In accordance with previousobservations that p66shc modulates oxidative stress in EPCsin response to high glucose [33] our results confirm thatAGEs induce increased p66shc expression in late EPCs viadownregulation of sirt1

5 Conclusions

The results of the present study together with the previousfindings indicate that HMGB-1 is not only an effector ofoxidative stress but also an inducer of oxidative stress in

diabetes-induced late EPCs In addition targeting HMGB-1with glycyrrhizin to inhibit oxidative stress is implicated asan attractive therapeutic strategy for impaired function of lateEPCs in DM

Disclosure

HanWu and Ran Li are co-first author

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Grants from the Natural ScienceFoundation of China (81070195 81200148 and 81270281)Jiangsu Key Laboratory for Molecular Medicine of NanjingUniversity Jiangsu Provincial Special Program of MedicalScience (BL2012014) State Key Laboratory of PharmaceuticalBiotechnology (KF-GN-200901) the Peak of Six Personnel inJiangsu Province (2013-WSN-008) Funds for DistinguishedYoung Scientists in Nanjing (Xie Jun) and the NaturalScience Foundation of Jiangsu Province (BK2010107)

References

[1] K-A Kim Y-J Shin J-H Kim et al ldquoDysfunction of endothe-lial progenitor cells under diabetic conditions and its underlyingmechanismsrdquo Archives of Pharmacal Research vol 35 no 2 pp223ndash234 2012

[2] D Tousoulis N Papageorgiou E Androulakis et al ldquoDia-betes mellitus-associated vascular impairment novel circulat-ing biomarkers and therapeutic approachesrdquo Journal of theAmerican College of Cardiology vol 62 no 8 pp 667ndash676 2013

[3] C Alev M Ii and T Asahara ldquoEndothelial progenitor cells anovel tool for the therapy of ischemic diseasesrdquo Antioxidantsand Redox Signaling vol 15 no 4 pp 949ndash965 2011

[4] A Georgescu N Alexandru A Constantinescu I TitorencuandD Popov ldquoThe promise of EPC-based therapies on vasculardysfunction in diabetesrdquo European Journal of Pharmacologyvol 669 no 1ndash3 pp 1ndash6 2011

[5] C J M Loomans E J P De Koning F J T Staal et alldquoEndothelial progenitor cell dysfunction a novel concept inthe pathogenesis of vascular complications of type 1 diabetesrdquoDiabetes vol 53 no 1 pp 195ndash199 2004

[6] N Antonio R Fernandes A Soares et al ldquoReduced levelsof circulating endothelial progenitor cells in acute myocardialinfarction patients with diabetes or pre-diabetes accompanyingthe glycemic continuumrdquo Cardiovascular Diabetology vol 13no 1 article 101 2014

[7] K Maiese ldquoNew insights for oxidative stress and diabetesmellitusrdquo Oxidative Medicine and Cellular Longevity vol 201517 pages 2015

[8] C J M Loomans E J P De Koning F J T Staal T JRabelink and A-J Van Zonneveld ldquoEndothelial progenitorcell dysfunction in type 1 diabetes another consequence ofoxidative stressrdquo Antioxidants and Redox Signaling vol 7 no11-12 pp 1468ndash1475 2005


[9] G P FadiniD Losordo and SDimmeler ldquoCritical reevaluationof endothelial progenitor cell phenotypes for therapeutic anddiagnostic userdquoCirculation Research vol 110 no 4 pp 624ndash6372012

[10] V L Bodiga S R Eda and S Bodiga ldquoAdvanced glycation endproducts role in pathology of diabetic cardiomyopathyrdquo HeartFailure Reviews vol 19 no 1 pp 49ndash63 2014

[11] M E Cooper ldquoImportance of advanced glycation end productsin diabetes-associated cardiovascular and renal diseaserdquo Amer-ican Journal of Hypertension vol 17 no 12 part 2 pp 31Sndash38S2004

[12] N M J Hanssen J W J Beulens S Van Dieren et al ldquoPlasmaadvanced glycation end products are associated with incidentcardiovascularevents in individuals with type 2 diabetes a Case-Cohort study with a median follow-Up of 10 years (EPIC-NL)rdquoDiabetes vol 64 no 1 pp 257ndash265 2015

[13] J Chen M Song S Yu et al ldquoAdvanced glycation endproductsalter functions and promote apoptosis in endothelial progenitorcells through receptor for advanced glycation endproductsmediate overpression of cell oxidant stressrdquo Molecular andCellular Biochemistry vol 335 no 1-2 pp 137ndash146 2010

[14] Q Chen L Dong L Wang L Kang and B Xu ldquoAdvancedglycation end products impair function of late endothelialprogenitor cells through effects on protein kinase Akt andcyclooxygenase-2rdquo Biochemical and Biophysical Research Com-munications vol 381 no 2 pp 192ndash197 2009

[15] Y Yu D Tang and R Kang ldquoOxidative stress-mediatedHMGB1 biologyrdquo Frontiers in Physiology vol 6 article 93 2015

[16] C Zhang M Mo W Ding et al ldquoHigh-mobility group box 1(HMGB1) impaired cardiac excitation-contraction coupling byenhancing the sarcoplasmic reticulum (SR) Ca2+ leak throughTLR4-ROS signaling in cardiomyocytesrdquo Journal of Molecularand Cellular Cardiology vol 74 pp 260ndash273 2014

[17] S EHermansen T Lund TKalstad K Ytrehus andTMyrmelldquoAdrenomedullin augments the angiogenic potential of lateoutgrowth endothelial progenitor cellsrdquo The American Journalof PhysiologymdashCell Physiology vol 300 no 4 pp C783ndashC7912011

[18] A Yamashita KNishihira YMatsuura et al ldquoPaucity of CD34-positive cells and increased expression of high-mobility groupbox 1 in coronary thrombus with type 2 diabetes mellitusrdquoAtherosclerosis vol 224 no 2 pp 511ndash514 2012

[19] F Delucchi R Berni C Frati et al ldquoResveratrol treatmentreduces cardiac progenitor cell dysfunction and preventsmorpho-functional ventricular remodeling in type-1 diabeticratsrdquo PLoS ONE vol 7 no 6 Article ID e39836 2012

[20] J Skrha M Kalousova J Svarcova et al ldquoRelationship ofsoluble RAGE and RAGE ligands HMGB1 and EN-RAGE toendothelial dysfunction in type 1 and type 2 diabetes mellitusrdquoExperimental and Clinical Endocrinology and Diabetes vol 120no 5 pp 277ndash281 2012

[21] H Wang H Qu and H Deng ldquoPlasma HMGB-1 levels insubjects with obesity and type 2 diabetes a cross-sectional studyin Chinardquo PLOS ONE vol 10 no 8 Article ID e0136564 2015

[22] D Yao and M Brownlee ldquoHyperglycemia-induced reactiveoxygen species increase expression of the receptor for advancedglycation end products (RAGE) and RAGE ligandsrdquo Diabetesvol 59 no 1 pp 249ndash255 2010

[23] Y Wang J Shan W Yang H Zheng and S Xue ldquoHighmobility group box 1 (HMGB1) mediates high-glucose-inducedcalcification in vascular smooth muscle cells of saphenousveinsrdquo Inflammation vol 36 no 6 pp 1592ndash1604 2013

[24] W-K Wang Q-H Lu J-N Zhang et al ldquoHMGB1 medi-ates hyperglycaemia-induced cardiomyocyte apoptosis viaERKEts-1 signalling pathwayrdquo Journal of Cellular andMolecularMedicine vol 18 no 11 pp 2311ndash2320 2015

[25] N Maugeri P Rovere-Querini M Baldini et al ldquoOxidativestress elicits plateletleukocyte inflammatory interactions viaHMGB1 a candidate formicrovessel injury in sytemic sclerosisrdquoAntioxidants amp Redox Signaling vol 20 no 7 pp 1060ndash10742014

[26] W Xu Y Lu J Yao et al ldquoNovel role of resveratrol suppres-sion of high-mobility group protein box 1 nucleocytoplasmictranslocation by the upregulation of sirtuin 1 in sepsis-inducedliver injuryrdquo Shock vol 42 no 5 pp 440ndash447 2014

[27] S Hamed B Brenner A Aharon D Daoud and A RoguinldquoNitric oxide and superoxide dismutase modulate endothelialprogenitor cell function in type 2 diabetes mellitusrdquo Cardiovas-cular Diabetology vol 8 article 56 2009

[28] M Kalousova and T Zima ldquoAGEs and RAGEmdashadvancedglycation end-products and their receptor in questions andanswersrdquo Vnitrni Lekarstvi vol 60 no 9 pp 720ndash724 2014

[29] J Chen L Huang M Song S Yu P Gao and J Jing ldquoC-reactive protein upregulates receptor for advanced glycationend products expression and alters antioxidant defenses inrat endothelial progenitor cellsrdquo Journal of CardiovascularPharmacology vol 53 no 5 pp 359ndash367 2009

[30] L Feng M Zhu M Zhang et al ldquoAmelioration of com-pound 441015840-diphenylmethane-bis(methyl)carbamate on highmobility group box1-mediated inflammation and oxidant stressresponses in human umbilical vein endothelial cells viaRAGEERK12NF-120581B pathwayrdquo International Immunophar-macology vol 15 no 2 pp 206ndash216 2013

[31] A Vikram Y-R Kim S Kumar et al ldquoCanonicalWnt signalinginduces vascular endothelial dysfunction via p66Shc-regulatedreactive oxygen speciesrdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 34 no 10 pp 2301ndash2309 2014

[32] S Zhou H-Z Chen Y-Z Wan et al ldquoRepression ofP66Shc expression by SIRT1 contributes to the prevention ofhyperglycemia-induced endothelial dysfunctionrdquo CirculationResearch vol 109 no 6 pp 639ndash648 2011

[33] V Di Stefano C Cencioni G Zaccagnini A Magenta MC Capogrossi and F Martelli ldquoP66ShcA modulates oxidativestress and survival of endothelial progenitor cells in response tohigh glucoserdquo Cardiovascular Research vol 82 no 3 pp 421ndash429 2009

[34] H Li X Zhang X Guan et al ldquoAdvanced glycation end prod-ucts impair the migration adhesion and secretion potentials oflate endothelial progenitor cellsrdquo Cardiovascular Diabetologyvol 11 article 46 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


rate and then contribute at least in part to the initiationand development of diabetic cardiovascular complications[10ndash12] In the past decade accumulating evidence hasshown that AGEs promote oxidative stress in EPCs and thenmediate EPC dysfunction in processes such as migrationtube formation and apoptosis [13 14]

High mobility group box-1 (HMGB-1) a nonchromo-somal nuclear protein is ubiquitously expressed in variouscells including monocytes cardiomyocytes and endothelialcells Once released into the serum in response to stressessuch as high glucose conditions HMGB-1 functions as aproinflammatory cytokine Recent studies have indicated thatoxidative stress promotes HMGB-1 release in various cells[15] resulting in the induction of reactive oxygen species(ROS) production in cardiomyocytes [16] However the roleof HMGB-1 in diabetes-induced oxidative stress in late EPCshas received little attention Thus we hypothesized thatdiabetes induces the release of HMGB-1 which subsequentlyenhances oxidative stress in late EPCs In the present studyHMGB-1 expression in serum and monocytes of diabeticmice was analyzed and the role of HMGB-1 in AGE-inducedoxidative stress in late EPCs was investigated in vitro

2 Methods

21 Induction and Assessment of Diabetes The experimentaland feeding protocols were approved and conducted in accor-dance with the laws and regulations controlling experimentson live animals in China and the Asian Convention forthe Protection of Vertebrate Animals used for Experimentaland Other Scientific Purposes Male C57BL6 mice werepurchased from the Model Animal Research Center of Nan-jing University (China) Before injection of streptozotocin(STZ) the mice were weighed and the blood glucose wasdetected in a sample obtained from the tail vein using bloodglucose monitoring system Diabetes was induced in mice byconsecutive intraperitoneal injections of STZ (40mgkgdaySigma-Aldrich USA) for 5 days After 3 days of STZ injectionblood glucose was determined as described previously Micewith blood glucose levels gt139mmoLl in three consecutivemeasurements were considered to be diabetic Mice treatedwith citrate buffer were used as nondiabetic controls Sixteenmice were divided into the following two groups normalnondiabetic group (N group 119899 = 8) and diabetic group (DMgroup 119899 = 8) Nonfasting blood glucose and weight weremeasured every month until the end of the experiment

22Measurement of SerumHMGB-1 Levels At the end of thisinvestigation the mice were anesthetized and sacrificed bydecapitation and blood was collected for serum separationSerum HMGB-1 concentrations were determined using amouse HMGB-1 ELISA kit (USCN Life Science China)according to the manufacturerrsquos instructions

23 Isolation of Monocytes from Mouse Bone Marrow Afterthe mice were sacrificed the femurs and tibias were immedi-ately excised and flushed with cold PBS After centrifugationat 1400 rpm for 5min the collected cells were filtered and

resuspended in red blood cell lysing buffer After 5min thecells were centrifuged at 1500 rpm for 5min After threewashing steps the monocytes were used in Western blotanalyses

24 EPC Isolation and Characterization The protocol wasapproved by the Ethical Committee of Institutional EthicsCommittee of Nanjing University Medical School (China)EPCs were isolated from the peripheral blood of healthyvolunteers as previously described [14 17] Briefly peripheralblood mononuclear cells (PBMCs) were collected by Ficoll-Paque PLUS (GE Healthcare Life Sciences USA) densitygradient centrifugation of peripheral blood PBMCs wereresuspended in endothelial cell growth medium-2 (EGM-2) (Lonza Switzerland) composed of endothelial cell basalmedium-2 (EBM-2) 5 fetal bovine serum (FBS) andgrowth factors containing vascular endothelial growth factor(VEGF) basic fibroblast growth factor (bFGF) epidermalgrowth factor (EGF) insulin-like factor-1 (IGF-1) hydrocor-tisone heparin and ascorbic acid After 4 days in culturemedium and nonadherent cells were removed the mediumwas replaced every 3 days After approximately 3 weeks theldquocobblestonerdquomorphology observed bymicroscope indicatedthat the cells were late EPCs These cells (passage lt 5) wereused in subsequent studies

To confirm the late EPC phenotype the cells were incu-bated with 5 120583gmL 11-dioctadecyl-3333-tetramethylin-docarbocyanine-labeled low density lipoprotein (Dil-acLDLMolecular Probes) for 2 h at 37∘C and then fixed with4 paraformaldehyde for 30min After being washed threetimes the cells were incubated with 10 120583gmL fluorescein-isothiocyanate-conjugated lectin (FITC-lectin Sigma USA)for 1 h at room temperature in the dark Additionally late EPCsurface markers were detected by immunocytochemistryusing FITC-CD34 (BD USA) allophycocyanin-KDR (APC-KDR RampD USA) and rabbit anti-CXCR4 (Abcam UK)Then goat anti-rabbit Alexa Fluor 488 was used to detect theexpression of CXCR4 Nuclei were stained with DAPI Thecells were observed by fluorescence microscopy (OlympusJapan)

25Western Blot Analysis After beingwashedwith cold PBSthe cells were resuspended in cell lysis buffer containing acocktail of protease inhibitors (1 100 Sigma USA) Aftercentrifugation at 15000 rpm for 10min the supernatant wascollected and stored at minus80∘C for later use The protein con-centration of the cell lysate was detected using a BCA proteinassay kit (Pierce USA) Total cell proteins or cell supernatantswere mixed with loading buffer and were heated in boilingwater for 10min The proteins were then separated by SDS-PAGE electrotransferred and blotted onto polyvinylidenedifluoride membranes The membranes were blocked with10 dried nonfat milk in 01 PBS-Tween-20 (PBST) for 2 hat room temperature and hybridized overnight at 4∘C withanti-HMGB-1 (1 1000 Bioworld Technology USA) anti-p66shc (1 1000 Santa Cruz USA) and anti-sirt1 (1 500Bioworld Technology USA) Membranes were then washedwith 01 PBST and incubated with appropriate secondary






3 Results







4 Discussion



Bloo

d gl

ucos

e (m

mol

L)

0 1 2 3Month

6

7

8

9

26

28

30

32

NDM

lowastlowast


(a)

0 1 2 3Month

Body

wei

ght (

g)

20

25

30

NDM

lowastlowast

lowastlowast

lowast

(b)

N DM

Seru

m H

MG

B-1

(pg

mL)

lowast

0

200

400

600

(c)N DM

120573-actin

HMGB-1H

MG

B-1120573

-act

in

lowast

00

05

10

15

20

(d)







BF CD34 KDR CXCR4

(a)

50 100 200 400

120573-actin

HMGB-1

HM

GB-

1120573

-act

in

00

05

10

15

20

25

BSA (400120583gmL)AGEs (120583gmL)


minus minus

+

lowastlowast


(b)

Cell supernatant

HMGB-1

HM

GB-

1120573

-act

in

0

1

2

3

4

50 100 200 400BSA (400120583gmL)


minus minus

+


(c)





sirt1

120573-actin

sirt1

120573-a

ctin

00

05

10

15

lowast

lowast

lowast

lowastlowast


+ + + +

Res (120583M) 10 20 40

(a)p6

6shc

120573-a

ctin

p66shc

120573-actin

00

05

10

15

lowastlowast

lowast

20

NS


minus minus minus

minus

+ + +

+ +

+

+ +

Res (10 120583M)Ex527 (10120583M)

(b)

DCFH-DA


(c)

HMGB-1

120573-actin

HM

GB-

1120573

-act

in

00

05

10

15

20

25




(d)





p66s

hc120573

-act

inp66shc

120573-actin


AGEs (100120583gmL) minus + + + +

00

05

10

15

20

lowast

(a)

SOD

activ

ity (

)


minus

minus

minus

+

50

+

100

+

200

+

lowast

0

50

100

150

(b)

sirt1

120573-actin

sirt1

120573-a

ctin


00

05

10

15

lowastlowast

(c)


119875 lt 001






RAGE

AGEHMGB-1

HMGB-1

Nucleus

Cytoplasm

Extracellular

p66shc uarr

sirt1 darr

ROS uarr




5 Conclusions



Disclosure




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results







4 Discussion



Bloo

d gl

ucos

e (m

mol

L)

0 1 2 3Month

6

7

8

9

26

28

30

32

NDM

lowastlowast


(a)

0 1 2 3Month

Body

wei

ght (

g)

20

25

30

NDM

lowastlowast

lowastlowast

lowast

(b)

N DM

Seru

m H

MG

B-1

(pg

mL)

lowast

0

200

400

600

(c)N DM

120573-actin

HMGB-1H

MG

B-1120573

-act

in

lowast

00

05

10

15

20

(d)







BF CD34 KDR CXCR4

(a)

50 100 200 400

120573-actin

HMGB-1

HM

GB-

1120573

-act

in

00

05

10

15

20

25

BSA (400120583gmL)AGEs (120583gmL)


minus minus

+

lowastlowast


(b)

Cell supernatant

HMGB-1

HM

GB-

1120573

-act

in

0

1

2

3

4

50 100 200 400BSA (400120583gmL)


minus minus

+


(c)





sirt1

120573-actin

sirt1

120573-a

ctin

00

05

10

15

lowast

lowast

lowast

lowastlowast


+ + + +

Res (120583M) 10 20 40

(a)p6

6shc

120573-a

ctin

p66shc

120573-actin

00

05

10

15

lowastlowast

lowast

20

NS


minus minus minus

minus

+ + +

+ +

+

+ +

Res (10 120583M)Ex527 (10120583M)

(b)

DCFH-DA


(c)

HMGB-1

120573-actin

HM

GB-

1120573

-act

in

00

05

10

15

20

25




(d)





p66s

hc120573

-act

inp66shc

120573-actin


AGEs (100120583gmL) minus + + + +

00

05

10

15

20

lowast

(a)

SOD

activ

ity (

)


minus

minus

minus

+

50

+

100

+

200

+

lowast

0

50

100

150

(b)

sirt1

120573-actin

sirt1

120573-a

ctin


00

05

10

15

lowastlowast

(c)


119875 lt 001






RAGE

AGEHMGB-1

HMGB-1

Nucleus

Cytoplasm

Extracellular

p66shc uarr

sirt1 darr

ROS uarr




5 Conclusions



Disclosure




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Bloo

d gl

ucos

e (m

mol

L)

0 1 2 3Month

6

7

8

9

26

28

30

32

NDM

lowastlowast


(a)

0 1 2 3Month

Body

wei

ght (

g)

20

25

30

NDM

lowastlowast

lowastlowast

lowast

(b)

N DM

Seru

m H

MG

B-1

(pg

mL)

lowast

0

200

400

600

(c)N DM

120573-actin

HMGB-1H

MG

B-1120573

-act

in

lowast

00

05

10

15

20

(d)







BF CD34 KDR CXCR4

(a)

50 100 200 400

120573-actin

HMGB-1

HM

GB-

1120573

-act

in

00

05

10

15

20

25

BSA (400120583gmL)AGEs (120583gmL)


minus minus

+

lowastlowast


(b)

Cell supernatant

HMGB-1

HM

GB-

1120573

-act

in

0

1

2

3

4

50 100 200 400BSA (400120583gmL)


minus minus

+


(c)





sirt1

120573-actin

sirt1

120573-a

ctin

00

05

10

15

lowast

lowast

lowast

lowastlowast


+ + + +

Res (120583M) 10 20 40

(a)p6

6shc

120573-a

ctin

p66shc

120573-actin

00

05

10

15

lowastlowast

lowast

20

NS


minus minus minus

minus

+ + +

+ +

+

+ +

Res (10 120583M)Ex527 (10120583M)

(b)

DCFH-DA


(c)

HMGB-1

120573-actin

HM

GB-

1120573

-act

in

00

05

10

15

20

25




(d)





p66s

hc120573

-act

inp66shc

120573-actin


AGEs (100120583gmL) minus + + + +

00

05

10

15

20

lowast

(a)

SOD

activ

ity (

)


minus

minus

minus

+

50

+

100

+

200

+

lowast

0

50

100

150

(b)

sirt1

120573-actin

sirt1

120573-a

ctin


00

05

10

15

lowastlowast

(c)


119875 lt 001






RAGE

AGEHMGB-1

HMGB-1

Nucleus

Cytoplasm

Extracellular

p66shc uarr

sirt1 darr

ROS uarr




5 Conclusions



Disclosure




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















BF CD34 KDR CXCR4

(a)

50 100 200 400

120573-actin

HMGB-1

HM

GB-

1120573

-act

in

00

05

10

15

20

25

BSA (400120583gmL)AGEs (120583gmL)


minus minus

+

lowastlowast


(b)

Cell supernatant

HMGB-1

HM

GB-

1120573

-act

in

0

1

2

3

4

50 100 200 400BSA (400120583gmL)


minus minus

+


(c)





sirt1

120573-actin

sirt1

120573-a

ctin

00

05

10

15

lowast

lowast

lowast

lowastlowast


+ + + +

Res (120583M) 10 20 40

(a)p6

6shc

120573-a

ctin

p66shc

120573-actin

00

05

10

15

lowastlowast

lowast

20

NS


minus minus minus

minus

+ + +

+ +

+

+ +

Res (10 120583M)Ex527 (10120583M)

(b)

DCFH-DA


(c)

HMGB-1

120573-actin

HM

GB-

1120573

-act

in

00

05

10

15

20

25




(d)





p66s

hc120573

-act

inp66shc

120573-actin


AGEs (100120583gmL) minus + + + +

00

05

10

15

20

lowast

(a)

SOD

activ

ity (

)


minus

minus

minus

+

50

+

100

+

200

+

lowast

0

50

100

150

(b)

sirt1

120573-actin

sirt1

120573-a

ctin


00

05

10

15

lowastlowast

(c)


119875 lt 001






RAGE

AGEHMGB-1

HMGB-1

Nucleus

Cytoplasm

Extracellular

p66shc uarr

sirt1 darr

ROS uarr




5 Conclusions



Disclosure




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















sirt1

120573-actin

sirt1

120573-a

ctin

00

05

10

15

lowast

lowast

lowast

lowastlowast


+ + + +

Res (120583M) 10 20 40

(a)p6

6shc

120573-a

ctin

p66shc

120573-actin

00

05

10

15

lowastlowast

lowast

20

NS


minus minus minus

minus

+ + +

+ +

+

+ +

Res (10 120583M)Ex527 (10120583M)

(b)

DCFH-DA


(c)

HMGB-1

120573-actin

HM

GB-

1120573

-act

in

00

05

10

15

20

25




(d)





p66s

hc120573

-act

inp66shc

120573-actin


AGEs (100120583gmL) minus + + + +

00

05

10

15

20

lowast

(a)

SOD

activ

ity (

)


minus

minus

minus

+

50

+

100

+

200

+

lowast

0

50

100

150

(b)

sirt1

120573-actin

sirt1

120573-a

ctin


00

05

10

15

lowastlowast

(c)


119875 lt 001






RAGE

AGEHMGB-1

HMGB-1

Nucleus

Cytoplasm

Extracellular

p66shc uarr

sirt1 darr

ROS uarr




5 Conclusions



Disclosure




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p66s

hc120573

-act

inp66shc

120573-actin


AGEs (100120583gmL) minus + + + +

00

05

10

15

20

lowast

(a)

SOD

activ

ity (

)


minus

minus

minus

+

50

+

100

+

200

+

lowast

0

50

100

150

(b)

sirt1

120573-actin

sirt1

120573-a

ctin


00

05

10

15

lowastlowast

(c)


119875 lt 001






RAGE

AGEHMGB-1

HMGB-1

Nucleus

Cytoplasm

Extracellular

p66shc uarr

sirt1 darr

ROS uarr




5 Conclusions



Disclosure




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















RAGE

AGEHMGB-1

HMGB-1

Nucleus

Cytoplasm

Extracellular

p66shc uarr

sirt1 darr

ROS uarr




5 Conclusions



Disclosure




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 Diabetes-Induced Oxidative Stress in...

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Transcript of Research Article Diabetes-Induced Oxidative Stress in...