Research ArticleUric Acid Induces Endothelial Dysfunction byActivating the HMGB1RAGE Signaling Pathway

Wei Cai12 Xi-Mei Duan13 Ying Liu1 Jiao Yu1 Yun-Liang Tang1 Ze-Lin Liu4 Shan Jiang1

Chun-Ping Zhang2 Jian-Ying Liu1 and Ji-Xiong Xu1

1Department of Endocrinology and Metabolism First Affiliated Hospital of Nanchang University Nanchang Jiangxi 330006 China2Department of Medical Genetics and Cell Biology Medical College of Nanchang University Nanchang Jiangxi 330006 China3Department of Endocrinology and Metabolism Peoplersquos Hospital of Shangrao City Shangrao Jiangxi 334600 China4Department of Endocrinology andMetabolism Fourth Affiliated Hospital of Nanchang University Nanchang Jiangxi 330003 China

Correspondence should be addressed to Ji-Xiong Xu xujixiong163com

Received 3 July 2016 Revised 25 October 2016 Accepted 6 November 2016 Published 1 January 2017

Academic Editor Senthil K Venugopal

Copyright copy 2017 Wei Cai et al This 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

Uric acid (UA) is a risk factor for endothelial dysfunction a process inwhich inflammationmay play an important role UA increaseshigh mobility group box chromosomal protein 1 (HMGB1) expression and extracellular release in endothelial cells HMGB1 isan inflammatory cytokine that interacts with the receptor for advanced glycation end products (RAGE) inducing an oxidativestress and inflammatory response which leads to endothelial dysfunction In this study human umbilical vein endothelial cells(HUVECs) were incubated with a high concentration of UA (20mgdL) after which endothelial function and the expression ofHMGB1 RAGE nuclear factor kappa B (NF-120581B) inflammatory cytokines and adhesion molecules were evaluated UA inhibitedendothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production inHUVECs increased intracellular HMGB1expression and extracellular HMGB1 secretion and upregulated RAGE expression UA also activated NF-120581B and increased thelevel of inflammatory cytokines Blocking RAGE significantly suppressed the upregulation of RAGE and HMGB1 and preventedthe increase in DNA binding activity of NF-120581B and the levels of inflammatory cytokines It also blocked the decrease in eNOSexpression and NO production induced by UA Our results suggest that high concentrations of UA cause endothelial dysfunctionvia the HMGB1RAGE signaling pathway

1 Introduction

Uric acid (UA) a final breakdown product of purinemetabolism is degraded by urate oxidase to allantoin andfreely excreted in the urine in most mammals [1] Howeverthe gene for urease in humans is a nonfunctioning pseu-dogene and overproduction or decreased excretion of UAresults in uniquely high level of serum UA and sometimeshyperuricemia [2ndash4] Hyperuricemia not only is intimatelyassociated with gout but also has a close connection withmany other diseases especially with cardiovascular disease[5ndash7] An elevated serum level of UA in humans is associatedwith systemic inflammation [8] endothelial dysfunction [9]hypertension [10] and cardiovascular disease [11] Manystudies have demonstrated that hyperuricemia is an indepen-dent risk factor for cardiovascular disease [12 13] It is well

known that hyperuricemia is one of the main risk factors forendothelial dysfunction [14 15] in which oxidative stress andinflammation may play an important role [16ndash18]

The receptor for advanced glycation end products(RAGE) a transmembrane multiligand receptor of theimmunoglobulin superfamily has been implicated in manychronic diseases [19 20] including atherosclerosis which isalso believed to be an inflammatory disorder [21] RAGEhas been linked with atherosclerosis due to its expressionon the surface of a wide variety of cells such as endothe-lial cells lymphocytes monocyte-derived macrophages andvascular smooth muscle cells which are implicated in thepathogenesis of atherosclerosis [22] In addition blockadeof RAGE signaling had significantly reduced progression ofatherosclerosis and the accumulation of RAGE-ligands wasalso reduced [23] The interaction of RAGE and its diverse

HindawiBioMed Research InternationalVolume 2017 Article ID 4391920 11 pageshttpsdoiorg10115520174391920

2 BioMed Research International

ligands such as advanced glycation end products (AGEs)some S100s amyloid peptide and high mobility group boxchromosomal protein 1 (HMGB1) stimulates oxidative stressgeneration and leads to cellular dysfunction [24] There isgrowing evidence to suggest that the RAGE-ligands axisplay an important role in the pathogenesis of cardiovasculardisease [25 26]

As a high affinity ligand of RAGE HMGB1 is a recentlydiscovered important extracellular mediator in systemicinflammation [27]HMGB1 is secreted as a latemediator witha delayed release during inflammation relative to classicalearly cytokines like tumor necrosis factor- (TNF-) 120572 HMGB1participates in the pathogenesis of systemic inflammationafter the early mediator response has resolved [28] HMGB1can be released by endothelial cells and mediates proin-flammatory responses of endothelial cells [29] High con-centrations of UA significantly increased mRNA expressionand extracellular release of HMGB1 from human umbilicalvein endothelial cells (HUVECs) [30] Extracellular HMGB1binding to RAGE activates nuclear factor kappa B (NF-120581B)which leads to proinflammation In this studywe investigatedwhether theHMGB1RAGE signaling pathway contributes toendothelial dysfunction induced by UA

2 Materials and Methods

21 Cell Culture HUVECs were obtained fromChina Centerfor Type Culture Collection Uric acid crystallites (Sigma LifeScience St Louis MO USA) were dissolved in 1N NaOHsolution HUVECs were cultured on gelatin-coated 25 cm2culture bottles and propagated in Dulbeccorsquos modified eaglemedium with 10 gL glucose supplemented with 10 heat-inactivated fetal bovine serum HUVECs were incubated at37∘C in a humidified atmosphere of 95 air and 5 carbondioxide The medium was refreshed every 2 to 3 days

22 UA Treatment HUVECs were cultured with 20mgdLUA solution for different times (0 12 24 and 48 h thezero time means the point of first incubation with UA)Inhibition of the interaction between HMGB1 and RAGEwas evaluated by using a specific RAGE blocking antibody(1 500 vv RampDSystemsMinneapolis USA)HUVECswerecultured with anti-RAGE antibody (5 120583gmL) for 2 h beforeUA (20mgdL) stimulation For controls HUVECs werecultured with control medium a normal goat IgG control(5 120583gmL) ormediumwith a control antibody (5 120583gmL) andUA (20mgdL)

23 RNA Extraction and Fluorogenic Quantitative PolymeraseChain Reaction Total RNA was extracted from HUVECsusing the TRIzol reagent (Invitrogen Carlsbad CA USA)according to the manufacturerrsquos instructions The RNAintegrity was checked by nucleic acid agarose gel elec-trophoresis and the RNA concentration was determinedby ultraviolet spectrophotometer Briefly 1 120583g of total RNAfor each sample was used to synthesize cDNA with anEasyScript First-Strand cDNA Synthesis SuperMix (Trans-Gen Biotech Beijing China) according to themanufacturerrsquos

protocol Fluorogenic quantitative PCR (FQ-PCR) was per-formed using the StepOneReal-Time PCR System (ThermoFisher Scientific Rockford IL) Primers were designed andsynthesized by Shanghai Sangon Biotech (Shanghai China)There were five pairs of primers The primer sequences forHMGB1 were 51015840-GGGATGGCAAAGTTTTTCCCTTTA-31015840 and 51015840-CACTAACCCTGCTGTTCGCT-31015840 For RAGEthe primer sequences were 51015840-GCTTGGAAGGTCCTG-TCTCC-31015840 and 51015840-CACGGACTCGGTAGTTGGAC-31015840 Forintercellular adhesion molecule- (ICAM-) 1 the primersequences were 51015840-CACAGTCACCTATGGCAACG-31015840 and51015840-GTGTCTCCTGGCTCTGGTTC-31015840 For vascular adhe-sion molecule- (VCAM-) 1 the primer sequences were 51015840-AGTTGAAGGATGCGGGAGTA-31015840 and 51015840-GTGTCTCCT-GGCTCTGGTTC-31015840 The mRNA expression of glyceralde-hyde 3-phosphate dehydrogenase (GAPDH) was used as aninternal reference

24 Western Blotting Assay RAGE endothelial nitric oxidesynthase (eNOS) ICAM-1 and VCAM-1 were examined incell samples by western blotting The extracellular HMGB1was concentrated using cellulose 3000molecular weight cut-off (MWCO) concentrating centrifugal filter units (MilliporeCorp Billerica MA USA) prior to western blotting NF-120581B was examined in nuclear and plasma protein samples bywestern blotting Nuclear protein and cytoplasmprotein frac-tionswere obtained using a nuclear plasma protein extractionkit (CWBio Beijing China) following the manufacturerrsquosinstructions The protein samples were dissolved in Laemmlibuffer boiled for 10min at 100∘C and equal amount ofproteins was separated on 10 SDS-PAGE gels (Bio-Rad Lab-oratories IncUSA)Themembranewas blocked in 5wtvolskim milk for 1 hour after the membrane was transferredto a nitrocellulose membrane (Cell Scientific) The proteinswere incubated with primary antibodies and followed withsecondary antibodies Polyclonal rabbit antibodies againstHMGB1 (Abcam Inc CambridgeMAUSA) RAGE (AbcamInc) NF-120581B p65 (Santa Cruz Biotechnology Santa CruzCA) eNOS (Abcam Inc) ICAM-1 (Affbiotech CincinnatiOH USA) VCAM-1 (BOSTER Wuhan Hubei China) pro-liferating cell nuclear antigen (PCNA Proteintech WuhanHubei China) and a monoclonal mouse antibody to 120573-actin (Affbiotech) were used as primary antibodies accord-ing to the manufacturersrsquo protocol (including dilutions)Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (ZSGB-BIO Beijing China)were usedTheproteinswere detected using enhanced chemi-luminescence reagents (Thermo Fisher Scientific RockfordIL) The images were captured through Gel Doc XR system(Bio-Rad Hercules CA USA) and analyzed using Image Labsoftware (vision 40) Anti-120573-actin antibody was used as theloading control for total proteins and cytoplasm protein andthe relative protein levels of nuclear protein were calculatedas densitometric ratios to PCNA

25 Measurement of Nitric Oxide Release Nitric oxide (NO)levels in cell culture supernatants were measured with Griessreagent by the nitrate reductase method using a nitric oxide

BioMed Research International 3

0

10

20

30

40 HUVECs supernatant

48h24h12h0h

lowast

lowastlowastlowastlowast

NO

(120583m

olL

)

(a)

eNOS

Actin

48h24h12h0h

00

05

10

15

Relat

ive u

nits

(nor

mal

ized

to ac

tin) HUVECs lysates

lowastlowast

lowastlowast

48h24h12h0h(b)

Figure 1 A high concentration of UA (20mgdL) induces endothelial dysfunction (a) UA significantly reduced NO release from HUVECsin a time-dependent manner (b) UA significantly reduced eNOS protein expression of HUVECs in a time-dependent manner Data areexpressed as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus 0 h group

assay kit (Nanjing Jiancheng Nanjing China) according tothe manufacturerrsquos protocol

26Measurement of Cytokines by Enzyme-Linked Immunosor-bent Assay (ELISA) Immunoreactive TNF-120572 and IL-6 weremeasured in duplicate using ELISA kits according to themanufacturerrsquos instructions (ExcellBio Shanghai China)

27 Statistical Analysis Data are expressed as means plusmnstandard deviation (SD) Differences among groups wereanalyzed by two-tailed Studentrsquos 119905-test or one-way ANOVAfollowed by post hoc Dunnettrsquos test as appropriate Allstatistical analyses were two-sided and a 119875 value of lessthan 005 was considered statistically significant Statisticalanalyses were carried out using SPSS version 17 (SPSS IncChicago IL USA)

3 Results

31 A High Concentration of UA-Induced Endothelial Dys-function To investigate whether a high concentration ofUA could induce endothelial dysfunction we detected thechanges in the amount of NO release and the expressioneNOS protein in HUVECs treated with 20mgdL UA fordifferent time periods When HUVECs were stimulated withUA for 24 h the amount of NO release was significantlyreduced versus control cells (119875 lt 005) (Figure 1(a)) as wasthe expression of eNOS protein (119875 lt 005) (Figure 1(b))These results show that a high concentration ofUAcan reducethe expression level of eNOS and the amount of NO releasedby HUVECs which leads to endothelial dysfunction

32 A High Concentration of UA Upregulates the Expressionof RAGE and HMGB1 in HUVECs Accompanied by anIncrease in Released HMGB1 To examine whether a highconcentration of UA can upregulate the expression of RAGEand HMGB1 we detected the mRNA and protein expressionof RAGE and HMGB1 by FQ-PCR and western blottingassay in HUVECs treated with 20mgdL UA When theHUVECs were stimulated with 20mgdL UA the mRNAexpression of RAGE and HMGB1 significantly increased ina time-dependent manner (Figure 2(a)) At the same timethe protein expression of RAGE gradually increased whilethe protein expression of HMGB1 decreased (Figure 2(b))Therefore in subsequent experiments the extracellular levelof HMGB1 was detected by western blotting assaysWe foundthat UA increased the extracellular level of HMGB1 in a time-dependent manner (Figure 2(c))The above results show thata high concentration of UA promotes the production andrelease of HMGB1 protein into the extracellular fluid

33 A High Concentration of UA Activates NF-120581B andInflammation Signals in HUVECs To clarify whether RAGEHMGB1 interactions can activate NF-120581B the expression ofNF-120581B p65 in the cytoplasm and nucleus of HUVECs wasdetected by western blotting assays After treatment with20mgdL UA we observed a decrease in cytoplasmic NF-120581B(Figure 3(a)) in a time-dependent manner

To examine whether UA induces inflammatory cytokinesand adhesion molecules which participate in the patho-genesis of atherosclerosis we detected the expression ofICAM-1 and VCAM-1 in HUVECs assessed by FQ-PCRand western blotting assays and the TNF-120572 and IL-6 release

4 BioMed Research International

HUVECs lysates

0

1

2

3

4

RAGEHMGB1

mRN

A (r

elat

ive u

nits)

48h24h12h0h

lowast

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(a)

RAGE

HMGB1

48h24h12h0h

120573-Actin

HUVECs lysates

00

05

10

15

20

lowast

lowast

lowastlowast

lowastlowast

RAGEHMGB1

48h24h12h0hProt

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

(b)

Medium

0

2

4

6

8

Relat

ive u

nits

48h24h12h0h

lowastlowast

lowastlowast

HMGB1

48h24h12h0h

(c)

Figure 2 A high concentration of UA (20mgdL) upregulates the expression of RAGE and HMGB1 in HUVECs accompanied by anincrease in releasedHMGB1 (a) UA significantly upregulated themRNA expression RAGE andHMGB1 in a time-dependentmanner (b) UAsignificantly upregulated the protein expression of RAGE in a time-dependent manner while the protein expression of HMGB1 decreased(c) UA significantly increased release of HMGB1 protein into the extracellular fluid Data are expressed as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001versus 0 h group

BioMed Research International 5

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

CytoplasmNuclear

Prot

ein

relat

ive u

nits

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

48h24h12h0h

48h24h12h0h

(a)

HUVECs lysates

0

2

4

6

8

10

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

lowast

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

48h24h12h0h

ICAM-1

VCAM-1

120573-Actin

48h24h12h0h

HUVECs lysates

0

1

2

3

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

lowast lowastlowast

lowast

lowastlowast

lowastlowast

48h24h12h0h

ICAM-1VCAM-1

(b)

Figure 3 Continued

6 BioMed Research International

HUVECs lysates

0 20 40 600

200

400

600

800

Time (hour)

IL-6

(pg

mL)

lowast

lowastlowast

lowastlowast

HUVECs lysates

0 20 40 600

100

200

300

400

Time (hour)

TNF-120572

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

(c)

Figure 3 A high concentration of UA (20mgdL) activates NF-120581B and inflammation signals in HUVECs (a) UA significantly decreasedthe expression of NF-120581B in the cytoplasm of HUVECs and significantly increased the expression of NF-120581B in the nucleus of HUVECs in atime-dependent manner (b) UA significantly increased the mRNA and protein expression of ICAM-1 and VCAM-1 in HUVECs in a time-dependent manner (c) UA significantly increased the levels of TNF-120572 and IL-6 release from HUVECs Data are expressed as means plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus 0 h group

from HUVECs measured by ELISA After treatment with20mgdL UA the mRNA and protein expressions of ICAM-1 and VCAM-1 in HUVECs significantly increased in atime-dependent manner (Figure 3(b)) and the TNF-120572 andIL-6 release from HUVECs also significantly increased ina time-dependent manner (Figure 3(c)) The above resultsshow that a high concentration of UA activates NF-120581Band inflammation signals in endothelial cells which has animportant role in the pathogenesis of atherosclerosis

34 Blockage of RAGE Suppresses Endothelial Dysfunctionand the HMGB1RAGE Signaling Pathway Induced by a HighConcentration of UA To investigate the role of RAGE inendothelial dysfunction induced by UA we used a specificantibody targeted against RAGE (anti-RAGE antibody) toneutralize RAGE Treatment of HUVECs with anti-RAGEantibody for 24 h significantly blocked the decrease in eNOSexpression and NO production induced by 20mgdL UA(Figures 4(a) and 4(b)) These results show that endothelialdysfunction induced by UA is partly mediated via RAGE

To further determine whether the expression of HMGB1and the activation of NF-120581B and inflammation signals inendothelial cells induced by UA were mediated by RAGEwe evaluated the effects of anti-RAGE antibody on theexpression of RAGE HMGB1 NF-120581B ICAM-1 and VCAM-1 and the release IL-6 and TNF-120572 from HUVECs Treatmentof HUVECs with anti-RAGE antibody for 24 h significantlysuppressed the upregulation of RAGEHMGB1 ICAM-1 andVCAM-1 (Figures 4(c) and 4(d)) prevented the increase inDNA binding activity of NF-120581B (Figure 4(e)) and decreasedthe release of IL-6 and TNF-120572 induced by 20mgdL UA (Fig-ure 4(f)) The above results show that the proinflammatoryactivity of a high concentration of UA is partly mediated viaRAGE in endothelial cells

4 Discussion

In this study we demonstrated a novel mechanism ofhigh concentration UA-induced endothelial dysfunction UAinhibited eNOS expression and NO release in endothelialcells and increased the levels of inflammatory cytokines bystimulating the HMGB1RAGE signaling pathway To ourknowledge this is the first time a link between UA-inducedendothelial dysfunction and the HMGB1RAGE signalingpathway has been established

Hyperuricemia not only is the etiological factor of gout[17 31] but also can lead to many different diseases [32]Many studies have demonstrated that hyperuricemia hasa close relationship with cardiovascular diseases and is anindependent risk factor for cardiovascular diseases [12 1333 34] It has been supposed that endothelial dysfunctionplays an important role in the initiation of atherosclerosis [35]and is an early marker for atherosclerosis [36] Many studieshave shown that oxidative stress and inflammatory responsesare the main pathogenesis of UA-induced vascular endothe-lial dysfunction [37ndash39] Hyperuricemia through inducingoxidative stress and inflammation reduces the expression ofeNOS and NO synthesis leading to damage of endothelialfunction [9] In the present study we also found that a highconcentration of UA downregulated the expression of eNOSand decreased the production of NO by HUVECs and alsoincreased the levels of inflammatory cytokines (IL-6 andTNF-120572) and adhesion molecules (ICAM-1 and VCAM-1)These results demonstrate that high concentrations of UAcan induce inflammatory responses and result in endothelialdysfunction However the exact mechanisms have not beendescribed yet

There is a large body of evidence to suggest that theRAGE-ligands axis has an important role in the pathogenesis

BioMed Research International 7

HUVECs lysates

00

05

10

15

24 hour

lowastlowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

1 2 3

eNOS

120573-Actin

4

(a)

HUVECs supernatant

0

10

20

30

40

24 hour

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControlN

O (120583

mol

L)

(b)

HUVECs lysates

0

2

4

6

8

10

RAGEHMGB1

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

24 hour

lowastlowast

lowastlowastlowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(c)

RAGE

HMGB1

ICAM-1

VCAM-1

1 2 3 4

120573-Actin

HUVECs lysates

00

05

10

15

20

24 hour

RAGEHMGB1

ICAM-1VCAM-1

lowast

lowast lowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

(d)

Figure 4 Continued

8 BioMed Research International

1 2 3 4

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

Prot

ein

relat

ive u

nits

24 hour

CytoplasmNuclear

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(e)

HUVECs supernatant

0

200

400

600

800

Con

cent

ratio

n (p

gm

L)

24 hour

IL-6TNF-120572

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(f)

Figure 4 Blockage of RAGE suppresses endothelial dysfunction and the HMGB1RAGE signaling pathway induced by a high concentrationof UA ((a) and (b)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly blocked the decrease in eNOS expression andNO production induced by 20mgdL UA ((c) and (d)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly suppressed theincrease in themRNA and protein expression of RAGE HMGB1 ICAM-1 and VCAM-1 induced by 20mgdLUA (e) Treatment of HUVECswith anti-RAGE antibody for 24 h significantly prevented the increase in DNA binding activity of NF-120581B (f) Treatment of HUVECs withanti-RAGE antibody for 24 h significantly decreased the release of IL-6 and TNF-120572 induced by 20mgdL UA Data are expressed as means plusmnSD lowast119875 lt 005 lowastlowast119875 lt 001 versus the untreated control and

119875 lt 005 versus the UA + control Ab

of atherosclerosis RAGE is a multiligand receptor of theimmunoglobulin superfamily which has been implicated inmany chronic inflammation diseases including atherosclero-sis [40] Previous people have found that diabetic apoEminusminusmice showed increased plaque area as well as increasedexpression of RAGE and its ligands but diabetic RAGEminusminusapoEminusminusmice showed significantly reduced plaque accumula-tion and expression of RAGE and its ligands [23]The studiesin vivo and in vitro have demonstrated that the interactionbetween RAGE and its ligands induces inflammation andcauses many chronic diseases including atherosclerosis [4142] As a high affinity ligand of RAGE HMGB1 belongs to thegroup of endogenous damage-associated molecular patternmolecules which are often associated with sterile inflam-mation [43] Extracellular HMGB1 participates in variouschronic diseases and HMGB1RAGE signaling pathway isone of themajor signaling pathways about these diseases [44]RAGE activation by extracellular HMGB1 leads to nucleartranslocation of NF-120581B in HUVECs and further promotesthe production and release of proinflammatory mediates and

contributes to the amplification of the inflammatory responseand finally induces endothelial dysfunction [45] Treatmentof HUVECs with a high concentration of UA results inincreased mRNA expression and extracellular release ofHMGB1 [30] which induces proinflammatory responses andendothelial dysfunction [29] Interactions between RAGEand HMGB1 stimulate oxidative stress and inflammatoryresponse leading to endothelial dysfunction [46 47] In thepresent study we found that a high concentration of UAincreased intracellular HMGB1 expression and extracellularsecretion while also increasing RAGE expression and acti-vating NF-120581B signaling in endothelial cells We also foundthat blocking (by anti-RAGE antibody) RAGE significantlysuppressed the upregulation of RAGE and HMGB1 andinhibited the increase in DNA binding activity of NF-120581Band the levels of inflammatory cytokines (IL-6 and TNF-120572) and adhesion molecules (ICAM-1 and VCAM-1) inducedby a high concentration of UA At the same time blockageof RAGE significantly prevented the decrease in the eNOSexpression and the NO production induced by a high

BioMed Research International 9

UA induces inflammation through HMGB1RAGE system

RAGE

Uric acid

HMGB1

ICAM VCAM

Inflammation

Anti-RAGEantibody

Endothelial cell

Nucleus

HMGB1

HMGB1

IL-6

NF-120581B

TNF-120572

NF-120581B

+

Figure 5 Potential mechanism of UA-induced inflammation in endothelial cell High uric acid stimulates the RAGE signaling pathwayand activates NF-120581B which results in the production and release of proinflammatory cytokines including the expression and extracellularrelease of HMGB1 in endothelial cells As a high affinity ligand of RAGE HMGB1 interacts with RAGE and contributes to the amplificationof the inflammatory response and finally induces endothelial dysfunction Blockade of RAGE by anti-RAGE antibody can suppress theHMGB1RAGE signaling pathway therefore alleviating endothelial dysfunction

concentration of UA These results imply that UA inducesendothelial dysfunction by stimulating the HMGB1RAGEsignaling pathway (Figure 5)

5 Conclusions

In conclusion the present study indicated that high con-centrations of UA induce endothelial dysfunction by theHMGB1RAGE signaling pathwayThese results provide newinsight into the mechanisms of UA-induced endothelialdysfunction and atherosclerosis

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Wei Cai and Xi-Mei Duan contributed equally to this work

Acknowledgments

The study was supported by grants from the National Nat-ural Science Funds of China (nos 81560154 81460018 and81160105) Jiangxi Provincial Science Technology Founda-tion of China (no 20151BBG70073) and Jiangxi ProvincialDepartment of Education Scientific Research Funds of China(nos GJJ13145 and GJJ13178)

References

[1] R J Johnson D-H Kang D Feig et al ldquoIs there a pathogeneticrole for uric acid in hypertension and cardiovascular and renaldiseaserdquo Hypertension vol 41 no 6 pp 1183ndash1190 2003

[2] V Vitart I Rudan C Hayward et al ldquoSLC2A9 is a newly iden-tified urate transporter influencing serum urate concentrationurate excretion and goutrdquo Nature Genetics vol 40 no 4 pp437ndash442 2008

[3] D I Feig D-H Kang T Nakagawa M Mazzali and R JJohnson ldquoUric acid and hypertensionrdquo Current HypertensionReports vol 8 no 2 pp 111ndash115 2006

[4] S Watanabe D-H Kang L L Feng et al ldquoUric acid hominoidevolution and the pathogenesis of salt-sensitivityrdquo Hyperten-sion vol 40 no 3 pp 355ndash360 2002

[5] M H Alderman ldquoUric acid and cardiovascular riskrdquo CurrentOpinion in Pharmacology vol 2 no 2 pp 126ndash130 2002

[6] C Zoccali R Maio F Mallamaci G Sesti and F PerticoneldquoUric acid and endothelial dysfunction in essential hyperten-sionrdquo Journal of theAmerican Society of Nephrology vol 17 no 5pp 1466ndash1471 2006

[7] E Agabiti-Rosei and G Grassi ldquoBeyond gout uric acid andcardiovascular diseasesrdquoCurrentMedical Research andOpinionvol 29 no 3 pp 33ndash39 2013

[8] S D Anker W Doehner M Rauchhaus et al ldquoUric acid andsurvival in chronic heart failure validation and application inmetabolic functional and hemodynamic stagingrdquo Circulationvol 107 no 15 pp 1991ndash1997 2003

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 3

0

10

20

30

40 HUVECs supernatant

48h24h12h0h

lowast

lowastlowastlowastlowast

NO

(120583m

olL

)

(a)

eNOS

Actin

48h24h12h0h

00

05

10

15

Relat

ive u

nits

(nor

mal

ized

to ac

tin) HUVECs lysates

lowastlowast

lowastlowast

48h24h12h0h(b)

Figure 1 A high concentration of UA (20mgdL) induces endothelial dysfunction (a) UA significantly reduced NO release from HUVECsin a time-dependent manner (b) UA significantly reduced eNOS protein expression of HUVECs in a time-dependent manner Data areexpressed as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus 0 h group

assay kit (Nanjing Jiancheng Nanjing China) according tothe manufacturerrsquos protocol

26Measurement of Cytokines by Enzyme-Linked Immunosor-bent Assay (ELISA) Immunoreactive TNF-120572 and IL-6 weremeasured in duplicate using ELISA kits according to themanufacturerrsquos instructions (ExcellBio Shanghai China)

27 Statistical Analysis Data are expressed as means plusmnstandard deviation (SD) Differences among groups wereanalyzed by two-tailed Studentrsquos 119905-test or one-way ANOVAfollowed by post hoc Dunnettrsquos test as appropriate Allstatistical analyses were two-sided and a 119875 value of lessthan 005 was considered statistically significant Statisticalanalyses were carried out using SPSS version 17 (SPSS IncChicago IL USA)

3 Results

31 A High Concentration of UA-Induced Endothelial Dys-function To investigate whether a high concentration ofUA could induce endothelial dysfunction we detected thechanges in the amount of NO release and the expressioneNOS protein in HUVECs treated with 20mgdL UA fordifferent time periods When HUVECs were stimulated withUA for 24 h the amount of NO release was significantlyreduced versus control cells (119875 lt 005) (Figure 1(a)) as wasthe expression of eNOS protein (119875 lt 005) (Figure 1(b))These results show that a high concentration ofUAcan reducethe expression level of eNOS and the amount of NO releasedby HUVECs which leads to endothelial dysfunction

32 A High Concentration of UA Upregulates the Expressionof RAGE and HMGB1 in HUVECs Accompanied by anIncrease in Released HMGB1 To examine whether a highconcentration of UA can upregulate the expression of RAGEand HMGB1 we detected the mRNA and protein expressionof RAGE and HMGB1 by FQ-PCR and western blottingassay in HUVECs treated with 20mgdL UA When theHUVECs were stimulated with 20mgdL UA the mRNAexpression of RAGE and HMGB1 significantly increased ina time-dependent manner (Figure 2(a)) At the same timethe protein expression of RAGE gradually increased whilethe protein expression of HMGB1 decreased (Figure 2(b))Therefore in subsequent experiments the extracellular levelof HMGB1 was detected by western blotting assaysWe foundthat UA increased the extracellular level of HMGB1 in a time-dependent manner (Figure 2(c))The above results show thata high concentration of UA promotes the production andrelease of HMGB1 protein into the extracellular fluid

33 A High Concentration of UA Activates NF-120581B andInflammation Signals in HUVECs To clarify whether RAGEHMGB1 interactions can activate NF-120581B the expression ofNF-120581B p65 in the cytoplasm and nucleus of HUVECs wasdetected by western blotting assays After treatment with20mgdL UA we observed a decrease in cytoplasmic NF-120581B(Figure 3(a)) in a time-dependent manner

To examine whether UA induces inflammatory cytokinesand adhesion molecules which participate in the patho-genesis of atherosclerosis we detected the expression ofICAM-1 and VCAM-1 in HUVECs assessed by FQ-PCRand western blotting assays and the TNF-120572 and IL-6 release

4 BioMed Research International

HUVECs lysates

0

1

2

3

4

RAGEHMGB1

mRN

A (r

elat

ive u

nits)

48h24h12h0h

lowast

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(a)

RAGE

HMGB1

48h24h12h0h

120573-Actin

HUVECs lysates

00

05

10

15

20

lowast

lowast

lowastlowast

lowastlowast

RAGEHMGB1

48h24h12h0hProt

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

(b)

Medium

0

2

4

6

8

Relat

ive u

nits

48h24h12h0h

lowastlowast

lowastlowast

HMGB1

48h24h12h0h

(c)

Figure 2 A high concentration of UA (20mgdL) upregulates the expression of RAGE and HMGB1 in HUVECs accompanied by anincrease in releasedHMGB1 (a) UA significantly upregulated themRNA expression RAGE andHMGB1 in a time-dependentmanner (b) UAsignificantly upregulated the protein expression of RAGE in a time-dependent manner while the protein expression of HMGB1 decreased(c) UA significantly increased release of HMGB1 protein into the extracellular fluid Data are expressed as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001versus 0 h group

BioMed Research International 5

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

CytoplasmNuclear

Prot

ein

relat

ive u

nits

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

48h24h12h0h

48h24h12h0h

(a)

HUVECs lysates

0

2

4

6

8

10

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

lowast

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

48h24h12h0h

ICAM-1

VCAM-1

120573-Actin

48h24h12h0h

HUVECs lysates

0

1

2

3

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

lowast lowastlowast

lowast

lowastlowast

lowastlowast

48h24h12h0h

ICAM-1VCAM-1

(b)

Figure 3 Continued

6 BioMed Research International

HUVECs lysates

0 20 40 600

200

400

600

800

Time (hour)

IL-6

(pg

mL)

lowast

lowastlowast

lowastlowast

HUVECs lysates

0 20 40 600

100

200

300

400

Time (hour)

TNF-120572

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

(c)

Figure 3 A high concentration of UA (20mgdL) activates NF-120581B and inflammation signals in HUVECs (a) UA significantly decreasedthe expression of NF-120581B in the cytoplasm of HUVECs and significantly increased the expression of NF-120581B in the nucleus of HUVECs in atime-dependent manner (b) UA significantly increased the mRNA and protein expression of ICAM-1 and VCAM-1 in HUVECs in a time-dependent manner (c) UA significantly increased the levels of TNF-120572 and IL-6 release from HUVECs Data are expressed as means plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus 0 h group

from HUVECs measured by ELISA After treatment with20mgdL UA the mRNA and protein expressions of ICAM-1 and VCAM-1 in HUVECs significantly increased in atime-dependent manner (Figure 3(b)) and the TNF-120572 andIL-6 release from HUVECs also significantly increased ina time-dependent manner (Figure 3(c)) The above resultsshow that a high concentration of UA activates NF-120581Band inflammation signals in endothelial cells which has animportant role in the pathogenesis of atherosclerosis

34 Blockage of RAGE Suppresses Endothelial Dysfunctionand the HMGB1RAGE Signaling Pathway Induced by a HighConcentration of UA To investigate the role of RAGE inendothelial dysfunction induced by UA we used a specificantibody targeted against RAGE (anti-RAGE antibody) toneutralize RAGE Treatment of HUVECs with anti-RAGEantibody for 24 h significantly blocked the decrease in eNOSexpression and NO production induced by 20mgdL UA(Figures 4(a) and 4(b)) These results show that endothelialdysfunction induced by UA is partly mediated via RAGE

To further determine whether the expression of HMGB1and the activation of NF-120581B and inflammation signals inendothelial cells induced by UA were mediated by RAGEwe evaluated the effects of anti-RAGE antibody on theexpression of RAGE HMGB1 NF-120581B ICAM-1 and VCAM-1 and the release IL-6 and TNF-120572 from HUVECs Treatmentof HUVECs with anti-RAGE antibody for 24 h significantlysuppressed the upregulation of RAGEHMGB1 ICAM-1 andVCAM-1 (Figures 4(c) and 4(d)) prevented the increase inDNA binding activity of NF-120581B (Figure 4(e)) and decreasedthe release of IL-6 and TNF-120572 induced by 20mgdL UA (Fig-ure 4(f)) The above results show that the proinflammatoryactivity of a high concentration of UA is partly mediated viaRAGE in endothelial cells

4 Discussion

In this study we demonstrated a novel mechanism ofhigh concentration UA-induced endothelial dysfunction UAinhibited eNOS expression and NO release in endothelialcells and increased the levels of inflammatory cytokines bystimulating the HMGB1RAGE signaling pathway To ourknowledge this is the first time a link between UA-inducedendothelial dysfunction and the HMGB1RAGE signalingpathway has been established

Hyperuricemia not only is the etiological factor of gout[17 31] but also can lead to many different diseases [32]Many studies have demonstrated that hyperuricemia hasa close relationship with cardiovascular diseases and is anindependent risk factor for cardiovascular diseases [12 1333 34] It has been supposed that endothelial dysfunctionplays an important role in the initiation of atherosclerosis [35]and is an early marker for atherosclerosis [36] Many studieshave shown that oxidative stress and inflammatory responsesare the main pathogenesis of UA-induced vascular endothe-lial dysfunction [37ndash39] Hyperuricemia through inducingoxidative stress and inflammation reduces the expression ofeNOS and NO synthesis leading to damage of endothelialfunction [9] In the present study we also found that a highconcentration of UA downregulated the expression of eNOSand decreased the production of NO by HUVECs and alsoincreased the levels of inflammatory cytokines (IL-6 andTNF-120572) and adhesion molecules (ICAM-1 and VCAM-1)These results demonstrate that high concentrations of UAcan induce inflammatory responses and result in endothelialdysfunction However the exact mechanisms have not beendescribed yet

There is a large body of evidence to suggest that theRAGE-ligands axis has an important role in the pathogenesis

BioMed Research International 7

HUVECs lysates

00

05

10

15

24 hour

lowastlowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

1 2 3

eNOS

120573-Actin

4

(a)

HUVECs supernatant

0

10

20

30

40

24 hour

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControlN

O (120583

mol

L)

(b)

HUVECs lysates

0

2

4

6

8

10

RAGEHMGB1

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

24 hour

lowastlowast

lowastlowastlowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(c)

RAGE

HMGB1

ICAM-1

VCAM-1

1 2 3 4

120573-Actin

HUVECs lysates

00

05

10

15

20

24 hour

RAGEHMGB1

ICAM-1VCAM-1

lowast

lowast lowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

(d)

Figure 4 Continued

8 BioMed Research International

1 2 3 4

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

Prot

ein

relat

ive u

nits

24 hour

CytoplasmNuclear

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(e)

HUVECs supernatant

0

200

400

600

800

Con

cent

ratio

n (p

gm

L)

24 hour

IL-6TNF-120572

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(f)

Figure 4 Blockage of RAGE suppresses endothelial dysfunction and the HMGB1RAGE signaling pathway induced by a high concentrationof UA ((a) and (b)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly blocked the decrease in eNOS expression andNO production induced by 20mgdL UA ((c) and (d)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly suppressed theincrease in themRNA and protein expression of RAGE HMGB1 ICAM-1 and VCAM-1 induced by 20mgdLUA (e) Treatment of HUVECswith anti-RAGE antibody for 24 h significantly prevented the increase in DNA binding activity of NF-120581B (f) Treatment of HUVECs withanti-RAGE antibody for 24 h significantly decreased the release of IL-6 and TNF-120572 induced by 20mgdL UA Data are expressed as means plusmnSD lowast119875 lt 005 lowastlowast119875 lt 001 versus the untreated control and

119875 lt 005 versus the UA + control Ab

of atherosclerosis RAGE is a multiligand receptor of theimmunoglobulin superfamily which has been implicated inmany chronic inflammation diseases including atherosclero-sis [40] Previous people have found that diabetic apoEminusminusmice showed increased plaque area as well as increasedexpression of RAGE and its ligands but diabetic RAGEminusminusapoEminusminusmice showed significantly reduced plaque accumula-tion and expression of RAGE and its ligands [23]The studiesin vivo and in vitro have demonstrated that the interactionbetween RAGE and its ligands induces inflammation andcauses many chronic diseases including atherosclerosis [4142] As a high affinity ligand of RAGE HMGB1 belongs to thegroup of endogenous damage-associated molecular patternmolecules which are often associated with sterile inflam-mation [43] Extracellular HMGB1 participates in variouschronic diseases and HMGB1RAGE signaling pathway isone of themajor signaling pathways about these diseases [44]RAGE activation by extracellular HMGB1 leads to nucleartranslocation of NF-120581B in HUVECs and further promotesthe production and release of proinflammatory mediates and

contributes to the amplification of the inflammatory responseand finally induces endothelial dysfunction [45] Treatmentof HUVECs with a high concentration of UA results inincreased mRNA expression and extracellular release ofHMGB1 [30] which induces proinflammatory responses andendothelial dysfunction [29] Interactions between RAGEand HMGB1 stimulate oxidative stress and inflammatoryresponse leading to endothelial dysfunction [46 47] In thepresent study we found that a high concentration of UAincreased intracellular HMGB1 expression and extracellularsecretion while also increasing RAGE expression and acti-vating NF-120581B signaling in endothelial cells We also foundthat blocking (by anti-RAGE antibody) RAGE significantlysuppressed the upregulation of RAGE and HMGB1 andinhibited the increase in DNA binding activity of NF-120581Band the levels of inflammatory cytokines (IL-6 and TNF-120572) and adhesion molecules (ICAM-1 and VCAM-1) inducedby a high concentration of UA At the same time blockageof RAGE significantly prevented the decrease in the eNOSexpression and the NO production induced by a high

BioMed Research International 9

UA induces inflammation through HMGB1RAGE system

RAGE

Uric acid

HMGB1

ICAM VCAM

Inflammation

Anti-RAGEantibody

Endothelial cell

Nucleus

HMGB1

HMGB1

IL-6

NF-120581B

TNF-120572

NF-120581B

+

Figure 5 Potential mechanism of UA-induced inflammation in endothelial cell High uric acid stimulates the RAGE signaling pathwayand activates NF-120581B which results in the production and release of proinflammatory cytokines including the expression and extracellularrelease of HMGB1 in endothelial cells As a high affinity ligand of RAGE HMGB1 interacts with RAGE and contributes to the amplificationof the inflammatory response and finally induces endothelial dysfunction Blockade of RAGE by anti-RAGE antibody can suppress theHMGB1RAGE signaling pathway therefore alleviating endothelial dysfunction

concentration of UA These results imply that UA inducesendothelial dysfunction by stimulating the HMGB1RAGEsignaling pathway (Figure 5)

5 Conclusions

In conclusion the present study indicated that high con-centrations of UA induce endothelial dysfunction by theHMGB1RAGE signaling pathwayThese results provide newinsight into the mechanisms of UA-induced endothelialdysfunction and atherosclerosis

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Wei Cai and Xi-Mei Duan contributed equally to this work

Acknowledgments

The study was supported by grants from the National Nat-ural Science Funds of China (nos 81560154 81460018 and81160105) Jiangxi Provincial Science Technology Founda-tion of China (no 20151BBG70073) and Jiangxi ProvincialDepartment of Education Scientific Research Funds of China(nos GJJ13145 and GJJ13178)

References

[1] R J Johnson D-H Kang D Feig et al ldquoIs there a pathogeneticrole for uric acid in hypertension and cardiovascular and renaldiseaserdquo Hypertension vol 41 no 6 pp 1183ndash1190 2003

[2] V Vitart I Rudan C Hayward et al ldquoSLC2A9 is a newly iden-tified urate transporter influencing serum urate concentrationurate excretion and goutrdquo Nature Genetics vol 40 no 4 pp437ndash442 2008

[3] D I Feig D-H Kang T Nakagawa M Mazzali and R JJohnson ldquoUric acid and hypertensionrdquo Current HypertensionReports vol 8 no 2 pp 111ndash115 2006

[4] S Watanabe D-H Kang L L Feng et al ldquoUric acid hominoidevolution and the pathogenesis of salt-sensitivityrdquo Hyperten-sion vol 40 no 3 pp 355ndash360 2002

[5] M H Alderman ldquoUric acid and cardiovascular riskrdquo CurrentOpinion in Pharmacology vol 2 no 2 pp 126ndash130 2002

[6] C Zoccali R Maio F Mallamaci G Sesti and F PerticoneldquoUric acid and endothelial dysfunction in essential hyperten-sionrdquo Journal of theAmerican Society of Nephrology vol 17 no 5pp 1466ndash1471 2006

[7] E Agabiti-Rosei and G Grassi ldquoBeyond gout uric acid andcardiovascular diseasesrdquoCurrentMedical Research andOpinionvol 29 no 3 pp 33ndash39 2013

[8] S D Anker W Doehner M Rauchhaus et al ldquoUric acid andsurvival in chronic heart failure validation and application inmetabolic functional and hemodynamic stagingrdquo Circulationvol 107 no 15 pp 1991ndash1997 2003

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 BioMed Research International

HUVECs lysates

0

1

2

3

4

RAGEHMGB1

mRN

A (r

elat

ive u

nits)

48h24h12h0h

lowast

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(a)

RAGE

HMGB1

48h24h12h0h

120573-Actin

HUVECs lysates

00

05

10

15

20

lowast

lowast

lowastlowast

lowastlowast

RAGEHMGB1

48h24h12h0hProt

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

(b)

Medium

0

2

4

6

8

Relat

ive u

nits

48h24h12h0h

lowastlowast

lowastlowast

HMGB1

48h24h12h0h

(c)

Figure 2 A high concentration of UA (20mgdL) upregulates the expression of RAGE and HMGB1 in HUVECs accompanied by anincrease in releasedHMGB1 (a) UA significantly upregulated themRNA expression RAGE andHMGB1 in a time-dependentmanner (b) UAsignificantly upregulated the protein expression of RAGE in a time-dependent manner while the protein expression of HMGB1 decreased(c) UA significantly increased release of HMGB1 protein into the extracellular fluid Data are expressed as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001versus 0 h group

BioMed Research International 5

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

CytoplasmNuclear

Prot

ein

relat

ive u

nits

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

48h24h12h0h

48h24h12h0h

(a)

HUVECs lysates

0

2

4

6

8

10

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

lowast

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

48h24h12h0h

ICAM-1

VCAM-1

120573-Actin

48h24h12h0h

HUVECs lysates

0

1

2

3

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

lowast lowastlowast

lowast

lowastlowast

lowastlowast

48h24h12h0h

ICAM-1VCAM-1

(b)

Figure 3 Continued

6 BioMed Research International

HUVECs lysates

0 20 40 600

200

400

600

800

Time (hour)

IL-6

(pg

mL)

lowast

lowastlowast

lowastlowast

HUVECs lysates

0 20 40 600

100

200

300

400

Time (hour)

TNF-120572

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

(c)

Figure 3 A high concentration of UA (20mgdL) activates NF-120581B and inflammation signals in HUVECs (a) UA significantly decreasedthe expression of NF-120581B in the cytoplasm of HUVECs and significantly increased the expression of NF-120581B in the nucleus of HUVECs in atime-dependent manner (b) UA significantly increased the mRNA and protein expression of ICAM-1 and VCAM-1 in HUVECs in a time-dependent manner (c) UA significantly increased the levels of TNF-120572 and IL-6 release from HUVECs Data are expressed as means plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus 0 h group

from HUVECs measured by ELISA After treatment with20mgdL UA the mRNA and protein expressions of ICAM-1 and VCAM-1 in HUVECs significantly increased in atime-dependent manner (Figure 3(b)) and the TNF-120572 andIL-6 release from HUVECs also significantly increased ina time-dependent manner (Figure 3(c)) The above resultsshow that a high concentration of UA activates NF-120581Band inflammation signals in endothelial cells which has animportant role in the pathogenesis of atherosclerosis

34 Blockage of RAGE Suppresses Endothelial Dysfunctionand the HMGB1RAGE Signaling Pathway Induced by a HighConcentration of UA To investigate the role of RAGE inendothelial dysfunction induced by UA we used a specificantibody targeted against RAGE (anti-RAGE antibody) toneutralize RAGE Treatment of HUVECs with anti-RAGEantibody for 24 h significantly blocked the decrease in eNOSexpression and NO production induced by 20mgdL UA(Figures 4(a) and 4(b)) These results show that endothelialdysfunction induced by UA is partly mediated via RAGE

To further determine whether the expression of HMGB1and the activation of NF-120581B and inflammation signals inendothelial cells induced by UA were mediated by RAGEwe evaluated the effects of anti-RAGE antibody on theexpression of RAGE HMGB1 NF-120581B ICAM-1 and VCAM-1 and the release IL-6 and TNF-120572 from HUVECs Treatmentof HUVECs with anti-RAGE antibody for 24 h significantlysuppressed the upregulation of RAGEHMGB1 ICAM-1 andVCAM-1 (Figures 4(c) and 4(d)) prevented the increase inDNA binding activity of NF-120581B (Figure 4(e)) and decreasedthe release of IL-6 and TNF-120572 induced by 20mgdL UA (Fig-ure 4(f)) The above results show that the proinflammatoryactivity of a high concentration of UA is partly mediated viaRAGE in endothelial cells

4 Discussion

In this study we demonstrated a novel mechanism ofhigh concentration UA-induced endothelial dysfunction UAinhibited eNOS expression and NO release in endothelialcells and increased the levels of inflammatory cytokines bystimulating the HMGB1RAGE signaling pathway To ourknowledge this is the first time a link between UA-inducedendothelial dysfunction and the HMGB1RAGE signalingpathway has been established

Hyperuricemia not only is the etiological factor of gout[17 31] but also can lead to many different diseases [32]Many studies have demonstrated that hyperuricemia hasa close relationship with cardiovascular diseases and is anindependent risk factor for cardiovascular diseases [12 1333 34] It has been supposed that endothelial dysfunctionplays an important role in the initiation of atherosclerosis [35]and is an early marker for atherosclerosis [36] Many studieshave shown that oxidative stress and inflammatory responsesare the main pathogenesis of UA-induced vascular endothe-lial dysfunction [37ndash39] Hyperuricemia through inducingoxidative stress and inflammation reduces the expression ofeNOS and NO synthesis leading to damage of endothelialfunction [9] In the present study we also found that a highconcentration of UA downregulated the expression of eNOSand decreased the production of NO by HUVECs and alsoincreased the levels of inflammatory cytokines (IL-6 andTNF-120572) and adhesion molecules (ICAM-1 and VCAM-1)These results demonstrate that high concentrations of UAcan induce inflammatory responses and result in endothelialdysfunction However the exact mechanisms have not beendescribed yet

There is a large body of evidence to suggest that theRAGE-ligands axis has an important role in the pathogenesis

BioMed Research International 7

HUVECs lysates

00

05

10

15

24 hour

lowastlowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

1 2 3

eNOS

120573-Actin

4

(a)

HUVECs supernatant

0

10

20

30

40

24 hour

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControlN

O (120583

mol

L)

(b)

HUVECs lysates

0

2

4

6

8

10

RAGEHMGB1

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

24 hour

lowastlowast

lowastlowastlowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(c)

RAGE

HMGB1

ICAM-1

VCAM-1

1 2 3 4

120573-Actin

HUVECs lysates

00

05

10

15

20

24 hour

RAGEHMGB1

ICAM-1VCAM-1

lowast

lowast lowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

(d)

Figure 4 Continued

8 BioMed Research International

1 2 3 4

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

Prot

ein

relat

ive u

nits

24 hour

CytoplasmNuclear

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(e)

HUVECs supernatant

0

200

400

600

800

Con

cent

ratio

n (p

gm

L)

24 hour

IL-6TNF-120572

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(f)

Figure 4 Blockage of RAGE suppresses endothelial dysfunction and the HMGB1RAGE signaling pathway induced by a high concentrationof UA ((a) and (b)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly blocked the decrease in eNOS expression andNO production induced by 20mgdL UA ((c) and (d)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly suppressed theincrease in themRNA and protein expression of RAGE HMGB1 ICAM-1 and VCAM-1 induced by 20mgdLUA (e) Treatment of HUVECswith anti-RAGE antibody for 24 h significantly prevented the increase in DNA binding activity of NF-120581B (f) Treatment of HUVECs withanti-RAGE antibody for 24 h significantly decreased the release of IL-6 and TNF-120572 induced by 20mgdL UA Data are expressed as means plusmnSD lowast119875 lt 005 lowastlowast119875 lt 001 versus the untreated control and

119875 lt 005 versus the UA + control Ab

of atherosclerosis RAGE is a multiligand receptor of theimmunoglobulin superfamily which has been implicated inmany chronic inflammation diseases including atherosclero-sis [40] Previous people have found that diabetic apoEminusminusmice showed increased plaque area as well as increasedexpression of RAGE and its ligands but diabetic RAGEminusminusapoEminusminusmice showed significantly reduced plaque accumula-tion and expression of RAGE and its ligands [23]The studiesin vivo and in vitro have demonstrated that the interactionbetween RAGE and its ligands induces inflammation andcauses many chronic diseases including atherosclerosis [4142] As a high affinity ligand of RAGE HMGB1 belongs to thegroup of endogenous damage-associated molecular patternmolecules which are often associated with sterile inflam-mation [43] Extracellular HMGB1 participates in variouschronic diseases and HMGB1RAGE signaling pathway isone of themajor signaling pathways about these diseases [44]RAGE activation by extracellular HMGB1 leads to nucleartranslocation of NF-120581B in HUVECs and further promotesthe production and release of proinflammatory mediates and

contributes to the amplification of the inflammatory responseand finally induces endothelial dysfunction [45] Treatmentof HUVECs with a high concentration of UA results inincreased mRNA expression and extracellular release ofHMGB1 [30] which induces proinflammatory responses andendothelial dysfunction [29] Interactions between RAGEand HMGB1 stimulate oxidative stress and inflammatoryresponse leading to endothelial dysfunction [46 47] In thepresent study we found that a high concentration of UAincreased intracellular HMGB1 expression and extracellularsecretion while also increasing RAGE expression and acti-vating NF-120581B signaling in endothelial cells We also foundthat blocking (by anti-RAGE antibody) RAGE significantlysuppressed the upregulation of RAGE and HMGB1 andinhibited the increase in DNA binding activity of NF-120581Band the levels of inflammatory cytokines (IL-6 and TNF-120572) and adhesion molecules (ICAM-1 and VCAM-1) inducedby a high concentration of UA At the same time blockageof RAGE significantly prevented the decrease in the eNOSexpression and the NO production induced by a high

BioMed Research International 9

UA induces inflammation through HMGB1RAGE system

RAGE

Uric acid

HMGB1

ICAM VCAM

Inflammation

Anti-RAGEantibody

Endothelial cell

Nucleus

HMGB1

HMGB1

IL-6

NF-120581B

TNF-120572

NF-120581B

+

Figure 5 Potential mechanism of UA-induced inflammation in endothelial cell High uric acid stimulates the RAGE signaling pathwayand activates NF-120581B which results in the production and release of proinflammatory cytokines including the expression and extracellularrelease of HMGB1 in endothelial cells As a high affinity ligand of RAGE HMGB1 interacts with RAGE and contributes to the amplificationof the inflammatory response and finally induces endothelial dysfunction Blockade of RAGE by anti-RAGE antibody can suppress theHMGB1RAGE signaling pathway therefore alleviating endothelial dysfunction

concentration of UA These results imply that UA inducesendothelial dysfunction by stimulating the HMGB1RAGEsignaling pathway (Figure 5)

5 Conclusions

In conclusion the present study indicated that high con-centrations of UA induce endothelial dysfunction by theHMGB1RAGE signaling pathwayThese results provide newinsight into the mechanisms of UA-induced endothelialdysfunction and atherosclerosis

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Wei Cai and Xi-Mei Duan contributed equally to this work

Acknowledgments

The study was supported by grants from the National Nat-ural Science Funds of China (nos 81560154 81460018 and81160105) Jiangxi Provincial Science Technology Founda-tion of China (no 20151BBG70073) and Jiangxi ProvincialDepartment of Education Scientific Research Funds of China(nos GJJ13145 and GJJ13178)

References

[1] R J Johnson D-H Kang D Feig et al ldquoIs there a pathogeneticrole for uric acid in hypertension and cardiovascular and renaldiseaserdquo Hypertension vol 41 no 6 pp 1183ndash1190 2003

[2] V Vitart I Rudan C Hayward et al ldquoSLC2A9 is a newly iden-tified urate transporter influencing serum urate concentrationurate excretion and goutrdquo Nature Genetics vol 40 no 4 pp437ndash442 2008

[3] D I Feig D-H Kang T Nakagawa M Mazzali and R JJohnson ldquoUric acid and hypertensionrdquo Current HypertensionReports vol 8 no 2 pp 111ndash115 2006

[4] S Watanabe D-H Kang L L Feng et al ldquoUric acid hominoidevolution and the pathogenesis of salt-sensitivityrdquo Hyperten-sion vol 40 no 3 pp 355ndash360 2002

[5] M H Alderman ldquoUric acid and cardiovascular riskrdquo CurrentOpinion in Pharmacology vol 2 no 2 pp 126ndash130 2002

[6] C Zoccali R Maio F Mallamaci G Sesti and F PerticoneldquoUric acid and endothelial dysfunction in essential hyperten-sionrdquo Journal of theAmerican Society of Nephrology vol 17 no 5pp 1466ndash1471 2006

[7] E Agabiti-Rosei and G Grassi ldquoBeyond gout uric acid andcardiovascular diseasesrdquoCurrentMedical Research andOpinionvol 29 no 3 pp 33ndash39 2013

[8] S D Anker W Doehner M Rauchhaus et al ldquoUric acid andsurvival in chronic heart failure validation and application inmetabolic functional and hemodynamic stagingrdquo Circulationvol 107 no 15 pp 1991ndash1997 2003

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 5

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

CytoplasmNuclear

Prot

ein

relat

ive u

nits

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

48h24h12h0h

48h24h12h0h

(a)

HUVECs lysates

0

2

4

6

8

10

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

lowast

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

48h24h12h0h

ICAM-1

VCAM-1

120573-Actin

48h24h12h0h

HUVECs lysates

0

1

2

3

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

lowast lowastlowast

lowast

lowastlowast

lowastlowast

48h24h12h0h

ICAM-1VCAM-1

(b)

Figure 3 Continued

6 BioMed Research International

HUVECs lysates

0 20 40 600

200

400

600

800

Time (hour)

IL-6

(pg

mL)

lowast

lowastlowast

lowastlowast

HUVECs lysates

0 20 40 600

100

200

300

400

Time (hour)

TNF-120572

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

(c)

Figure 3 A high concentration of UA (20mgdL) activates NF-120581B and inflammation signals in HUVECs (a) UA significantly decreasedthe expression of NF-120581B in the cytoplasm of HUVECs and significantly increased the expression of NF-120581B in the nucleus of HUVECs in atime-dependent manner (b) UA significantly increased the mRNA and protein expression of ICAM-1 and VCAM-1 in HUVECs in a time-dependent manner (c) UA significantly increased the levels of TNF-120572 and IL-6 release from HUVECs Data are expressed as means plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus 0 h group

from HUVECs measured by ELISA After treatment with20mgdL UA the mRNA and protein expressions of ICAM-1 and VCAM-1 in HUVECs significantly increased in atime-dependent manner (Figure 3(b)) and the TNF-120572 andIL-6 release from HUVECs also significantly increased ina time-dependent manner (Figure 3(c)) The above resultsshow that a high concentration of UA activates NF-120581Band inflammation signals in endothelial cells which has animportant role in the pathogenesis of atherosclerosis

34 Blockage of RAGE Suppresses Endothelial Dysfunctionand the HMGB1RAGE Signaling Pathway Induced by a HighConcentration of UA To investigate the role of RAGE inendothelial dysfunction induced by UA we used a specificantibody targeted against RAGE (anti-RAGE antibody) toneutralize RAGE Treatment of HUVECs with anti-RAGEantibody for 24 h significantly blocked the decrease in eNOSexpression and NO production induced by 20mgdL UA(Figures 4(a) and 4(b)) These results show that endothelialdysfunction induced by UA is partly mediated via RAGE

To further determine whether the expression of HMGB1and the activation of NF-120581B and inflammation signals inendothelial cells induced by UA were mediated by RAGEwe evaluated the effects of anti-RAGE antibody on theexpression of RAGE HMGB1 NF-120581B ICAM-1 and VCAM-1 and the release IL-6 and TNF-120572 from HUVECs Treatmentof HUVECs with anti-RAGE antibody for 24 h significantlysuppressed the upregulation of RAGEHMGB1 ICAM-1 andVCAM-1 (Figures 4(c) and 4(d)) prevented the increase inDNA binding activity of NF-120581B (Figure 4(e)) and decreasedthe release of IL-6 and TNF-120572 induced by 20mgdL UA (Fig-ure 4(f)) The above results show that the proinflammatoryactivity of a high concentration of UA is partly mediated viaRAGE in endothelial cells

4 Discussion

In this study we demonstrated a novel mechanism ofhigh concentration UA-induced endothelial dysfunction UAinhibited eNOS expression and NO release in endothelialcells and increased the levels of inflammatory cytokines bystimulating the HMGB1RAGE signaling pathway To ourknowledge this is the first time a link between UA-inducedendothelial dysfunction and the HMGB1RAGE signalingpathway has been established

Hyperuricemia not only is the etiological factor of gout[17 31] but also can lead to many different diseases [32]Many studies have demonstrated that hyperuricemia hasa close relationship with cardiovascular diseases and is anindependent risk factor for cardiovascular diseases [12 1333 34] It has been supposed that endothelial dysfunctionplays an important role in the initiation of atherosclerosis [35]and is an early marker for atherosclerosis [36] Many studieshave shown that oxidative stress and inflammatory responsesare the main pathogenesis of UA-induced vascular endothe-lial dysfunction [37ndash39] Hyperuricemia through inducingoxidative stress and inflammation reduces the expression ofeNOS and NO synthesis leading to damage of endothelialfunction [9] In the present study we also found that a highconcentration of UA downregulated the expression of eNOSand decreased the production of NO by HUVECs and alsoincreased the levels of inflammatory cytokines (IL-6 andTNF-120572) and adhesion molecules (ICAM-1 and VCAM-1)These results demonstrate that high concentrations of UAcan induce inflammatory responses and result in endothelialdysfunction However the exact mechanisms have not beendescribed yet

There is a large body of evidence to suggest that theRAGE-ligands axis has an important role in the pathogenesis

BioMed Research International 7

HUVECs lysates

00

05

10

15

24 hour

lowastlowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

1 2 3

eNOS

120573-Actin

4

(a)

HUVECs supernatant

0

10

20

30

40

24 hour

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControlN

O (120583

mol

L)

(b)

HUVECs lysates

0

2

4

6

8

10

RAGEHMGB1

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

24 hour

lowastlowast

lowastlowastlowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(c)

RAGE

HMGB1

ICAM-1

VCAM-1

1 2 3 4

120573-Actin

HUVECs lysates

00

05

10

15

20

24 hour

RAGEHMGB1

ICAM-1VCAM-1

lowast

lowast lowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

(d)

Figure 4 Continued

8 BioMed Research International

1 2 3 4

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

Prot

ein

relat

ive u

nits

24 hour

CytoplasmNuclear

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(e)

HUVECs supernatant

0

200

400

600

800

Con

cent

ratio

n (p

gm

L)

24 hour

IL-6TNF-120572

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(f)

Figure 4 Blockage of RAGE suppresses endothelial dysfunction and the HMGB1RAGE signaling pathway induced by a high concentrationof UA ((a) and (b)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly blocked the decrease in eNOS expression andNO production induced by 20mgdL UA ((c) and (d)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly suppressed theincrease in themRNA and protein expression of RAGE HMGB1 ICAM-1 and VCAM-1 induced by 20mgdLUA (e) Treatment of HUVECswith anti-RAGE antibody for 24 h significantly prevented the increase in DNA binding activity of NF-120581B (f) Treatment of HUVECs withanti-RAGE antibody for 24 h significantly decreased the release of IL-6 and TNF-120572 induced by 20mgdL UA Data are expressed as means plusmnSD lowast119875 lt 005 lowastlowast119875 lt 001 versus the untreated control and

119875 lt 005 versus the UA + control Ab

of atherosclerosis RAGE is a multiligand receptor of theimmunoglobulin superfamily which has been implicated inmany chronic inflammation diseases including atherosclero-sis [40] Previous people have found that diabetic apoEminusminusmice showed increased plaque area as well as increasedexpression of RAGE and its ligands but diabetic RAGEminusminusapoEminusminusmice showed significantly reduced plaque accumula-tion and expression of RAGE and its ligands [23]The studiesin vivo and in vitro have demonstrated that the interactionbetween RAGE and its ligands induces inflammation andcauses many chronic diseases including atherosclerosis [4142] As a high affinity ligand of RAGE HMGB1 belongs to thegroup of endogenous damage-associated molecular patternmolecules which are often associated with sterile inflam-mation [43] Extracellular HMGB1 participates in variouschronic diseases and HMGB1RAGE signaling pathway isone of themajor signaling pathways about these diseases [44]RAGE activation by extracellular HMGB1 leads to nucleartranslocation of NF-120581B in HUVECs and further promotesthe production and release of proinflammatory mediates and

contributes to the amplification of the inflammatory responseand finally induces endothelial dysfunction [45] Treatmentof HUVECs with a high concentration of UA results inincreased mRNA expression and extracellular release ofHMGB1 [30] which induces proinflammatory responses andendothelial dysfunction [29] Interactions between RAGEand HMGB1 stimulate oxidative stress and inflammatoryresponse leading to endothelial dysfunction [46 47] In thepresent study we found that a high concentration of UAincreased intracellular HMGB1 expression and extracellularsecretion while also increasing RAGE expression and acti-vating NF-120581B signaling in endothelial cells We also foundthat blocking (by anti-RAGE antibody) RAGE significantlysuppressed the upregulation of RAGE and HMGB1 andinhibited the increase in DNA binding activity of NF-120581Band the levels of inflammatory cytokines (IL-6 and TNF-120572) and adhesion molecules (ICAM-1 and VCAM-1) inducedby a high concentration of UA At the same time blockageof RAGE significantly prevented the decrease in the eNOSexpression and the NO production induced by a high

BioMed Research International 9

UA induces inflammation through HMGB1RAGE system

RAGE

Uric acid

HMGB1

ICAM VCAM

Inflammation

Anti-RAGEantibody

Endothelial cell

Nucleus

HMGB1

HMGB1

IL-6

NF-120581B

TNF-120572

NF-120581B

+

Figure 5 Potential mechanism of UA-induced inflammation in endothelial cell High uric acid stimulates the RAGE signaling pathwayand activates NF-120581B which results in the production and release of proinflammatory cytokines including the expression and extracellularrelease of HMGB1 in endothelial cells As a high affinity ligand of RAGE HMGB1 interacts with RAGE and contributes to the amplificationof the inflammatory response and finally induces endothelial dysfunction Blockade of RAGE by anti-RAGE antibody can suppress theHMGB1RAGE signaling pathway therefore alleviating endothelial dysfunction

concentration of UA These results imply that UA inducesendothelial dysfunction by stimulating the HMGB1RAGEsignaling pathway (Figure 5)

5 Conclusions

In conclusion the present study indicated that high con-centrations of UA induce endothelial dysfunction by theHMGB1RAGE signaling pathwayThese results provide newinsight into the mechanisms of UA-induced endothelialdysfunction and atherosclerosis

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Wei Cai and Xi-Mei Duan contributed equally to this work

Acknowledgments

The study was supported by grants from the National Nat-ural Science Funds of China (nos 81560154 81460018 and81160105) Jiangxi Provincial Science Technology Founda-tion of China (no 20151BBG70073) and Jiangxi ProvincialDepartment of Education Scientific Research Funds of China(nos GJJ13145 and GJJ13178)

References

[1] R J Johnson D-H Kang D Feig et al ldquoIs there a pathogeneticrole for uric acid in hypertension and cardiovascular and renaldiseaserdquo Hypertension vol 41 no 6 pp 1183ndash1190 2003

[2] V Vitart I Rudan C Hayward et al ldquoSLC2A9 is a newly iden-tified urate transporter influencing serum urate concentrationurate excretion and goutrdquo Nature Genetics vol 40 no 4 pp437ndash442 2008

[3] D I Feig D-H Kang T Nakagawa M Mazzali and R JJohnson ldquoUric acid and hypertensionrdquo Current HypertensionReports vol 8 no 2 pp 111ndash115 2006

[4] S Watanabe D-H Kang L L Feng et al ldquoUric acid hominoidevolution and the pathogenesis of salt-sensitivityrdquo Hyperten-sion vol 40 no 3 pp 355ndash360 2002

[5] M H Alderman ldquoUric acid and cardiovascular riskrdquo CurrentOpinion in Pharmacology vol 2 no 2 pp 126ndash130 2002

[6] C Zoccali R Maio F Mallamaci G Sesti and F PerticoneldquoUric acid and endothelial dysfunction in essential hyperten-sionrdquo Journal of theAmerican Society of Nephrology vol 17 no 5pp 1466ndash1471 2006

[7] E Agabiti-Rosei and G Grassi ldquoBeyond gout uric acid andcardiovascular diseasesrdquoCurrentMedical Research andOpinionvol 29 no 3 pp 33ndash39 2013

[8] S D Anker W Doehner M Rauchhaus et al ldquoUric acid andsurvival in chronic heart failure validation and application inmetabolic functional and hemodynamic stagingrdquo Circulationvol 107 no 15 pp 1991ndash1997 2003

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 BioMed Research International

HUVECs lysates

0 20 40 600

200

400

600

800

Time (hour)

IL-6

(pg

mL)

lowast

lowastlowast

lowastlowast

HUVECs lysates

0 20 40 600

100

200

300

400

Time (hour)

TNF-120572

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

(c)

Figure 3 A high concentration of UA (20mgdL) activates NF-120581B and inflammation signals in HUVECs (a) UA significantly decreasedthe expression of NF-120581B in the cytoplasm of HUVECs and significantly increased the expression of NF-120581B in the nucleus of HUVECs in atime-dependent manner (b) UA significantly increased the mRNA and protein expression of ICAM-1 and VCAM-1 in HUVECs in a time-dependent manner (c) UA significantly increased the levels of TNF-120572 and IL-6 release from HUVECs Data are expressed as means plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus 0 h group

from HUVECs measured by ELISA After treatment with20mgdL UA the mRNA and protein expressions of ICAM-1 and VCAM-1 in HUVECs significantly increased in atime-dependent manner (Figure 3(b)) and the TNF-120572 andIL-6 release from HUVECs also significantly increased ina time-dependent manner (Figure 3(c)) The above resultsshow that a high concentration of UA activates NF-120581Band inflammation signals in endothelial cells which has animportant role in the pathogenesis of atherosclerosis

34 Blockage of RAGE Suppresses Endothelial Dysfunctionand the HMGB1RAGE Signaling Pathway Induced by a HighConcentration of UA To investigate the role of RAGE inendothelial dysfunction induced by UA we used a specificantibody targeted against RAGE (anti-RAGE antibody) toneutralize RAGE Treatment of HUVECs with anti-RAGEantibody for 24 h significantly blocked the decrease in eNOSexpression and NO production induced by 20mgdL UA(Figures 4(a) and 4(b)) These results show that endothelialdysfunction induced by UA is partly mediated via RAGE

To further determine whether the expression of HMGB1and the activation of NF-120581B and inflammation signals inendothelial cells induced by UA were mediated by RAGEwe evaluated the effects of anti-RAGE antibody on theexpression of RAGE HMGB1 NF-120581B ICAM-1 and VCAM-1 and the release IL-6 and TNF-120572 from HUVECs Treatmentof HUVECs with anti-RAGE antibody for 24 h significantlysuppressed the upregulation of RAGEHMGB1 ICAM-1 andVCAM-1 (Figures 4(c) and 4(d)) prevented the increase inDNA binding activity of NF-120581B (Figure 4(e)) and decreasedthe release of IL-6 and TNF-120572 induced by 20mgdL UA (Fig-ure 4(f)) The above results show that the proinflammatoryactivity of a high concentration of UA is partly mediated viaRAGE in endothelial cells

4 Discussion

In this study we demonstrated a novel mechanism ofhigh concentration UA-induced endothelial dysfunction UAinhibited eNOS expression and NO release in endothelialcells and increased the levels of inflammatory cytokines bystimulating the HMGB1RAGE signaling pathway To ourknowledge this is the first time a link between UA-inducedendothelial dysfunction and the HMGB1RAGE signalingpathway has been established

Hyperuricemia not only is the etiological factor of gout[17 31] but also can lead to many different diseases [32]Many studies have demonstrated that hyperuricemia hasa close relationship with cardiovascular diseases and is anindependent risk factor for cardiovascular diseases [12 1333 34] It has been supposed that endothelial dysfunctionplays an important role in the initiation of atherosclerosis [35]and is an early marker for atherosclerosis [36] Many studieshave shown that oxidative stress and inflammatory responsesare the main pathogenesis of UA-induced vascular endothe-lial dysfunction [37ndash39] Hyperuricemia through inducingoxidative stress and inflammation reduces the expression ofeNOS and NO synthesis leading to damage of endothelialfunction [9] In the present study we also found that a highconcentration of UA downregulated the expression of eNOSand decreased the production of NO by HUVECs and alsoincreased the levels of inflammatory cytokines (IL-6 andTNF-120572) and adhesion molecules (ICAM-1 and VCAM-1)These results demonstrate that high concentrations of UAcan induce inflammatory responses and result in endothelialdysfunction However the exact mechanisms have not beendescribed yet

There is a large body of evidence to suggest that theRAGE-ligands axis has an important role in the pathogenesis

BioMed Research International 7

HUVECs lysates

00

05

10

15

24 hour

lowastlowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

1 2 3

eNOS

120573-Actin

4

(a)

HUVECs supernatant

0

10

20

30

40

24 hour

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControlN

O (120583

mol

L)

(b)

HUVECs lysates

0

2

4

6

8

10

RAGEHMGB1

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

24 hour

lowastlowast

lowastlowastlowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(c)

RAGE

HMGB1

ICAM-1

VCAM-1

1 2 3 4

120573-Actin

HUVECs lysates

00

05

10

15

20

24 hour

RAGEHMGB1

ICAM-1VCAM-1

lowast

lowast lowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

(d)

Figure 4 Continued

8 BioMed Research International

1 2 3 4

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

Prot

ein

relat

ive u

nits

24 hour

CytoplasmNuclear

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(e)

HUVECs supernatant

0

200

400

600

800

Con

cent

ratio

n (p

gm

L)

24 hour

IL-6TNF-120572

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(f)

Figure 4 Blockage of RAGE suppresses endothelial dysfunction and the HMGB1RAGE signaling pathway induced by a high concentrationof UA ((a) and (b)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly blocked the decrease in eNOS expression andNO production induced by 20mgdL UA ((c) and (d)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly suppressed theincrease in themRNA and protein expression of RAGE HMGB1 ICAM-1 and VCAM-1 induced by 20mgdLUA (e) Treatment of HUVECswith anti-RAGE antibody for 24 h significantly prevented the increase in DNA binding activity of NF-120581B (f) Treatment of HUVECs withanti-RAGE antibody for 24 h significantly decreased the release of IL-6 and TNF-120572 induced by 20mgdL UA Data are expressed as means plusmnSD lowast119875 lt 005 lowastlowast119875 lt 001 versus the untreated control and

119875 lt 005 versus the UA + control Ab

of atherosclerosis RAGE is a multiligand receptor of theimmunoglobulin superfamily which has been implicated inmany chronic inflammation diseases including atherosclero-sis [40] Previous people have found that diabetic apoEminusminusmice showed increased plaque area as well as increasedexpression of RAGE and its ligands but diabetic RAGEminusminusapoEminusminusmice showed significantly reduced plaque accumula-tion and expression of RAGE and its ligands [23]The studiesin vivo and in vitro have demonstrated that the interactionbetween RAGE and its ligands induces inflammation andcauses many chronic diseases including atherosclerosis [4142] As a high affinity ligand of RAGE HMGB1 belongs to thegroup of endogenous damage-associated molecular patternmolecules which are often associated with sterile inflam-mation [43] Extracellular HMGB1 participates in variouschronic diseases and HMGB1RAGE signaling pathway isone of themajor signaling pathways about these diseases [44]RAGE activation by extracellular HMGB1 leads to nucleartranslocation of NF-120581B in HUVECs and further promotesthe production and release of proinflammatory mediates and

contributes to the amplification of the inflammatory responseand finally induces endothelial dysfunction [45] Treatmentof HUVECs with a high concentration of UA results inincreased mRNA expression and extracellular release ofHMGB1 [30] which induces proinflammatory responses andendothelial dysfunction [29] Interactions between RAGEand HMGB1 stimulate oxidative stress and inflammatoryresponse leading to endothelial dysfunction [46 47] In thepresent study we found that a high concentration of UAincreased intracellular HMGB1 expression and extracellularsecretion while also increasing RAGE expression and acti-vating NF-120581B signaling in endothelial cells We also foundthat blocking (by anti-RAGE antibody) RAGE significantlysuppressed the upregulation of RAGE and HMGB1 andinhibited the increase in DNA binding activity of NF-120581Band the levels of inflammatory cytokines (IL-6 and TNF-120572) and adhesion molecules (ICAM-1 and VCAM-1) inducedby a high concentration of UA At the same time blockageof RAGE significantly prevented the decrease in the eNOSexpression and the NO production induced by a high

BioMed Research International 9

UA induces inflammation through HMGB1RAGE system

RAGE

Uric acid

HMGB1

ICAM VCAM

Inflammation

Anti-RAGEantibody

Endothelial cell

Nucleus

HMGB1

HMGB1

IL-6

NF-120581B

TNF-120572

NF-120581B

+

Figure 5 Potential mechanism of UA-induced inflammation in endothelial cell High uric acid stimulates the RAGE signaling pathwayand activates NF-120581B which results in the production and release of proinflammatory cytokines including the expression and extracellularrelease of HMGB1 in endothelial cells As a high affinity ligand of RAGE HMGB1 interacts with RAGE and contributes to the amplificationof the inflammatory response and finally induces endothelial dysfunction Blockade of RAGE by anti-RAGE antibody can suppress theHMGB1RAGE signaling pathway therefore alleviating endothelial dysfunction

concentration of UA These results imply that UA inducesendothelial dysfunction by stimulating the HMGB1RAGEsignaling pathway (Figure 5)

5 Conclusions

In conclusion the present study indicated that high con-centrations of UA induce endothelial dysfunction by theHMGB1RAGE signaling pathwayThese results provide newinsight into the mechanisms of UA-induced endothelialdysfunction and atherosclerosis

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Wei Cai and Xi-Mei Duan contributed equally to this work

Acknowledgments

The study was supported by grants from the National Nat-ural Science Funds of China (nos 81560154 81460018 and81160105) Jiangxi Provincial Science Technology Founda-tion of China (no 20151BBG70073) and Jiangxi ProvincialDepartment of Education Scientific Research Funds of China(nos GJJ13145 and GJJ13178)

References

[1] R J Johnson D-H Kang D Feig et al ldquoIs there a pathogeneticrole for uric acid in hypertension and cardiovascular and renaldiseaserdquo Hypertension vol 41 no 6 pp 1183ndash1190 2003

[2] V Vitart I Rudan C Hayward et al ldquoSLC2A9 is a newly iden-tified urate transporter influencing serum urate concentrationurate excretion and goutrdquo Nature Genetics vol 40 no 4 pp437ndash442 2008

[3] D I Feig D-H Kang T Nakagawa M Mazzali and R JJohnson ldquoUric acid and hypertensionrdquo Current HypertensionReports vol 8 no 2 pp 111ndash115 2006

[4] S Watanabe D-H Kang L L Feng et al ldquoUric acid hominoidevolution and the pathogenesis of salt-sensitivityrdquo Hyperten-sion vol 40 no 3 pp 355ndash360 2002

[5] M H Alderman ldquoUric acid and cardiovascular riskrdquo CurrentOpinion in Pharmacology vol 2 no 2 pp 126ndash130 2002

[6] C Zoccali R Maio F Mallamaci G Sesti and F PerticoneldquoUric acid and endothelial dysfunction in essential hyperten-sionrdquo Journal of theAmerican Society of Nephrology vol 17 no 5pp 1466ndash1471 2006

[7] E Agabiti-Rosei and G Grassi ldquoBeyond gout uric acid andcardiovascular diseasesrdquoCurrentMedical Research andOpinionvol 29 no 3 pp 33ndash39 2013

[8] S D Anker W Doehner M Rauchhaus et al ldquoUric acid andsurvival in chronic heart failure validation and application inmetabolic functional and hemodynamic stagingrdquo Circulationvol 107 no 15 pp 1991ndash1997 2003

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 7

HUVECs lysates

00

05

10

15

24 hour

lowastlowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

1 2 3

eNOS

120573-Actin

4

(a)

HUVECs supernatant

0

10

20

30

40

24 hour

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControlN

O (120583

mol

L)

(b)

HUVECs lysates

0

2

4

6

8

10

RAGEHMGB1

ICAM-1VCAM-1

mRN

A (r

elat

ive u

nits)

24 hour

lowastlowast

lowastlowastlowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(c)

RAGE

HMGB1

ICAM-1

VCAM-1

1 2 3 4

120573-Actin

HUVECs lysates

00

05

10

15

20

24 hour

RAGEHMGB1

ICAM-1VCAM-1

lowast

lowast lowast

Prot

ein

relat

ive u

nits

(nor

mal

ized

to120573

-act

in)

UA +anti-RAGE

UA +control Ab

Control AbControl

(d)

Figure 4 Continued

8 BioMed Research International

1 2 3 4

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

Prot

ein

relat

ive u

nits

24 hour

CytoplasmNuclear

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(e)

HUVECs supernatant

0

200

400

600

800

Con

cent

ratio

n (p

gm

L)

24 hour

IL-6TNF-120572

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(f)

Figure 4 Blockage of RAGE suppresses endothelial dysfunction and the HMGB1RAGE signaling pathway induced by a high concentrationof UA ((a) and (b)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly blocked the decrease in eNOS expression andNO production induced by 20mgdL UA ((c) and (d)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly suppressed theincrease in themRNA and protein expression of RAGE HMGB1 ICAM-1 and VCAM-1 induced by 20mgdLUA (e) Treatment of HUVECswith anti-RAGE antibody for 24 h significantly prevented the increase in DNA binding activity of NF-120581B (f) Treatment of HUVECs withanti-RAGE antibody for 24 h significantly decreased the release of IL-6 and TNF-120572 induced by 20mgdL UA Data are expressed as means plusmnSD lowast119875 lt 005 lowastlowast119875 lt 001 versus the untreated control and

119875 lt 005 versus the UA + control Ab

of atherosclerosis RAGE is a multiligand receptor of theimmunoglobulin superfamily which has been implicated inmany chronic inflammation diseases including atherosclero-sis [40] Previous people have found that diabetic apoEminusminusmice showed increased plaque area as well as increasedexpression of RAGE and its ligands but diabetic RAGEminusminusapoEminusminusmice showed significantly reduced plaque accumula-tion and expression of RAGE and its ligands [23]The studiesin vivo and in vitro have demonstrated that the interactionbetween RAGE and its ligands induces inflammation andcauses many chronic diseases including atherosclerosis [4142] As a high affinity ligand of RAGE HMGB1 belongs to thegroup of endogenous damage-associated molecular patternmolecules which are often associated with sterile inflam-mation [43] Extracellular HMGB1 participates in variouschronic diseases and HMGB1RAGE signaling pathway isone of themajor signaling pathways about these diseases [44]RAGE activation by extracellular HMGB1 leads to nucleartranslocation of NF-120581B in HUVECs and further promotesthe production and release of proinflammatory mediates and

contributes to the amplification of the inflammatory responseand finally induces endothelial dysfunction [45] Treatmentof HUVECs with a high concentration of UA results inincreased mRNA expression and extracellular release ofHMGB1 [30] which induces proinflammatory responses andendothelial dysfunction [29] Interactions between RAGEand HMGB1 stimulate oxidative stress and inflammatoryresponse leading to endothelial dysfunction [46 47] In thepresent study we found that a high concentration of UAincreased intracellular HMGB1 expression and extracellularsecretion while also increasing RAGE expression and acti-vating NF-120581B signaling in endothelial cells We also foundthat blocking (by anti-RAGE antibody) RAGE significantlysuppressed the upregulation of RAGE and HMGB1 andinhibited the increase in DNA binding activity of NF-120581Band the levels of inflammatory cytokines (IL-6 and TNF-120572) and adhesion molecules (ICAM-1 and VCAM-1) inducedby a high concentration of UA At the same time blockageof RAGE significantly prevented the decrease in the eNOSexpression and the NO production induced by a high

BioMed Research International 9

UA induces inflammation through HMGB1RAGE system

RAGE

Uric acid

HMGB1

ICAM VCAM

Inflammation

Anti-RAGEantibody

Endothelial cell

Nucleus

HMGB1

HMGB1

IL-6

NF-120581B

TNF-120572

NF-120581B

+

Figure 5 Potential mechanism of UA-induced inflammation in endothelial cell High uric acid stimulates the RAGE signaling pathwayand activates NF-120581B which results in the production and release of proinflammatory cytokines including the expression and extracellularrelease of HMGB1 in endothelial cells As a high affinity ligand of RAGE HMGB1 interacts with RAGE and contributes to the amplificationof the inflammatory response and finally induces endothelial dysfunction Blockade of RAGE by anti-RAGE antibody can suppress theHMGB1RAGE signaling pathway therefore alleviating endothelial dysfunction

concentration of UA These results imply that UA inducesendothelial dysfunction by stimulating the HMGB1RAGEsignaling pathway (Figure 5)

5 Conclusions

In conclusion the present study indicated that high con-centrations of UA induce endothelial dysfunction by theHMGB1RAGE signaling pathwayThese results provide newinsight into the mechanisms of UA-induced endothelialdysfunction and atherosclerosis

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Wei Cai and Xi-Mei Duan contributed equally to this work

Acknowledgments

The study was supported by grants from the National Nat-ural Science Funds of China (nos 81560154 81460018 and81160105) Jiangxi Provincial Science Technology Founda-tion of China (no 20151BBG70073) and Jiangxi ProvincialDepartment of Education Scientific Research Funds of China(nos GJJ13145 and GJJ13178)

References

[1] R J Johnson D-H Kang D Feig et al ldquoIs there a pathogeneticrole for uric acid in hypertension and cardiovascular and renaldiseaserdquo Hypertension vol 41 no 6 pp 1183ndash1190 2003

[2] V Vitart I Rudan C Hayward et al ldquoSLC2A9 is a newly iden-tified urate transporter influencing serum urate concentrationurate excretion and goutrdquo Nature Genetics vol 40 no 4 pp437ndash442 2008

[3] D I Feig D-H Kang T Nakagawa M Mazzali and R JJohnson ldquoUric acid and hypertensionrdquo Current HypertensionReports vol 8 no 2 pp 111ndash115 2006

[4] S Watanabe D-H Kang L L Feng et al ldquoUric acid hominoidevolution and the pathogenesis of salt-sensitivityrdquo Hyperten-sion vol 40 no 3 pp 355ndash360 2002

[5] M H Alderman ldquoUric acid and cardiovascular riskrdquo CurrentOpinion in Pharmacology vol 2 no 2 pp 126ndash130 2002

[6] C Zoccali R Maio F Mallamaci G Sesti and F PerticoneldquoUric acid and endothelial dysfunction in essential hyperten-sionrdquo Journal of theAmerican Society of Nephrology vol 17 no 5pp 1466ndash1471 2006

[7] E Agabiti-Rosei and G Grassi ldquoBeyond gout uric acid andcardiovascular diseasesrdquoCurrentMedical Research andOpinionvol 29 no 3 pp 33ndash39 2013

[8] S D Anker W Doehner M Rauchhaus et al ldquoUric acid andsurvival in chronic heart failure validation and application inmetabolic functional and hemodynamic stagingrdquo Circulationvol 107 no 15 pp 1991ndash1997 2003

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 BioMed Research International

1 2 3 4

PCNA

NF-120581B (N)

NF-120581B (C)

120573-Actin

00

05

10

15

20

25

Prot

ein

relat

ive u

nits

24 hour

CytoplasmNuclear

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(e)

HUVECs supernatant

0

200

400

600

800

Con

cent

ratio

n (p

gm

L)

24 hour

IL-6TNF-120572

lowastlowast

lowastlowast

UA +anti-RAGE

UA +control Ab

Control AbControl

(f)

Figure 4 Blockage of RAGE suppresses endothelial dysfunction and the HMGB1RAGE signaling pathway induced by a high concentrationof UA ((a) and (b)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly blocked the decrease in eNOS expression andNO production induced by 20mgdL UA ((c) and (d)) Treatment of HUVECs with anti-RAGE antibody for 24 h significantly suppressed theincrease in themRNA and protein expression of RAGE HMGB1 ICAM-1 and VCAM-1 induced by 20mgdLUA (e) Treatment of HUVECswith anti-RAGE antibody for 24 h significantly prevented the increase in DNA binding activity of NF-120581B (f) Treatment of HUVECs withanti-RAGE antibody for 24 h significantly decreased the release of IL-6 and TNF-120572 induced by 20mgdL UA Data are expressed as means plusmnSD lowast119875 lt 005 lowastlowast119875 lt 001 versus the untreated control and

119875 lt 005 versus the UA + control Ab

of atherosclerosis RAGE is a multiligand receptor of theimmunoglobulin superfamily which has been implicated inmany chronic inflammation diseases including atherosclero-sis [40] Previous people have found that diabetic apoEminusminusmice showed increased plaque area as well as increasedexpression of RAGE and its ligands but diabetic RAGEminusminusapoEminusminusmice showed significantly reduced plaque accumula-tion and expression of RAGE and its ligands [23]The studiesin vivo and in vitro have demonstrated that the interactionbetween RAGE and its ligands induces inflammation andcauses many chronic diseases including atherosclerosis [4142] As a high affinity ligand of RAGE HMGB1 belongs to thegroup of endogenous damage-associated molecular patternmolecules which are often associated with sterile inflam-mation [43] Extracellular HMGB1 participates in variouschronic diseases and HMGB1RAGE signaling pathway isone of themajor signaling pathways about these diseases [44]RAGE activation by extracellular HMGB1 leads to nucleartranslocation of NF-120581B in HUVECs and further promotesthe production and release of proinflammatory mediates and

contributes to the amplification of the inflammatory responseand finally induces endothelial dysfunction [45] Treatmentof HUVECs with a high concentration of UA results inincreased mRNA expression and extracellular release ofHMGB1 [30] which induces proinflammatory responses andendothelial dysfunction [29] Interactions between RAGEand HMGB1 stimulate oxidative stress and inflammatoryresponse leading to endothelial dysfunction [46 47] In thepresent study we found that a high concentration of UAincreased intracellular HMGB1 expression and extracellularsecretion while also increasing RAGE expression and acti-vating NF-120581B signaling in endothelial cells We also foundthat blocking (by anti-RAGE antibody) RAGE significantlysuppressed the upregulation of RAGE and HMGB1 andinhibited the increase in DNA binding activity of NF-120581Band the levels of inflammatory cytokines (IL-6 and TNF-120572) and adhesion molecules (ICAM-1 and VCAM-1) inducedby a high concentration of UA At the same time blockageof RAGE significantly prevented the decrease in the eNOSexpression and the NO production induced by a high

BioMed Research International 9

UA induces inflammation through HMGB1RAGE system

RAGE

Uric acid

HMGB1

ICAM VCAM

Inflammation

Anti-RAGEantibody

Endothelial cell

Nucleus

HMGB1

HMGB1

IL-6

NF-120581B

TNF-120572

NF-120581B

+

Figure 5 Potential mechanism of UA-induced inflammation in endothelial cell High uric acid stimulates the RAGE signaling pathwayand activates NF-120581B which results in the production and release of proinflammatory cytokines including the expression and extracellularrelease of HMGB1 in endothelial cells As a high affinity ligand of RAGE HMGB1 interacts with RAGE and contributes to the amplificationof the inflammatory response and finally induces endothelial dysfunction Blockade of RAGE by anti-RAGE antibody can suppress theHMGB1RAGE signaling pathway therefore alleviating endothelial dysfunction

concentration of UA These results imply that UA inducesendothelial dysfunction by stimulating the HMGB1RAGEsignaling pathway (Figure 5)

5 Conclusions

In conclusion the present study indicated that high con-centrations of UA induce endothelial dysfunction by theHMGB1RAGE signaling pathwayThese results provide newinsight into the mechanisms of UA-induced endothelialdysfunction and atherosclerosis

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Wei Cai and Xi-Mei Duan contributed equally to this work

Acknowledgments

The study was supported by grants from the National Nat-ural Science Funds of China (nos 81560154 81460018 and81160105) Jiangxi Provincial Science Technology Founda-tion of China (no 20151BBG70073) and Jiangxi ProvincialDepartment of Education Scientific Research Funds of China(nos GJJ13145 and GJJ13178)

References

[1] R J Johnson D-H Kang D Feig et al ldquoIs there a pathogeneticrole for uric acid in hypertension and cardiovascular and renaldiseaserdquo Hypertension vol 41 no 6 pp 1183ndash1190 2003

[2] V Vitart I Rudan C Hayward et al ldquoSLC2A9 is a newly iden-tified urate transporter influencing serum urate concentrationurate excretion and goutrdquo Nature Genetics vol 40 no 4 pp437ndash442 2008

[3] D I Feig D-H Kang T Nakagawa M Mazzali and R JJohnson ldquoUric acid and hypertensionrdquo Current HypertensionReports vol 8 no 2 pp 111ndash115 2006

[4] S Watanabe D-H Kang L L Feng et al ldquoUric acid hominoidevolution and the pathogenesis of salt-sensitivityrdquo Hyperten-sion vol 40 no 3 pp 355ndash360 2002

[5] M H Alderman ldquoUric acid and cardiovascular riskrdquo CurrentOpinion in Pharmacology vol 2 no 2 pp 126ndash130 2002

[6] C Zoccali R Maio F Mallamaci G Sesti and F PerticoneldquoUric acid and endothelial dysfunction in essential hyperten-sionrdquo Journal of theAmerican Society of Nephrology vol 17 no 5pp 1466ndash1471 2006

[7] E Agabiti-Rosei and G Grassi ldquoBeyond gout uric acid andcardiovascular diseasesrdquoCurrentMedical Research andOpinionvol 29 no 3 pp 33ndash39 2013

[8] S D Anker W Doehner M Rauchhaus et al ldquoUric acid andsurvival in chronic heart failure validation and application inmetabolic functional and hemodynamic stagingrdquo Circulationvol 107 no 15 pp 1991ndash1997 2003

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 9

UA induces inflammation through HMGB1RAGE system

RAGE

Uric acid

HMGB1

ICAM VCAM

Inflammation

Anti-RAGEantibody

Endothelial cell

Nucleus

HMGB1

HMGB1

IL-6

NF-120581B

TNF-120572

NF-120581B

+

Figure 5 Potential mechanism of UA-induced inflammation in endothelial cell High uric acid stimulates the RAGE signaling pathwayand activates NF-120581B which results in the production and release of proinflammatory cytokines including the expression and extracellularrelease of HMGB1 in endothelial cells As a high affinity ligand of RAGE HMGB1 interacts with RAGE and contributes to the amplificationof the inflammatory response and finally induces endothelial dysfunction Blockade of RAGE by anti-RAGE antibody can suppress theHMGB1RAGE signaling pathway therefore alleviating endothelial dysfunction

concentration of UA These results imply that UA inducesendothelial dysfunction by stimulating the HMGB1RAGEsignaling pathway (Figure 5)

5 Conclusions

In conclusion the present study indicated that high con-centrations of UA induce endothelial dysfunction by theHMGB1RAGE signaling pathwayThese results provide newinsight into the mechanisms of UA-induced endothelialdysfunction and atherosclerosis

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Wei Cai and Xi-Mei Duan contributed equally to this work

Acknowledgments

The study was supported by grants from the National Nat-ural Science Funds of China (nos 81560154 81460018 and81160105) Jiangxi Provincial Science Technology Founda-tion of China (no 20151BBG70073) and Jiangxi ProvincialDepartment of Education Scientific Research Funds of China(nos GJJ13145 and GJJ13178)

References

[1] R J Johnson D-H Kang D Feig et al ldquoIs there a pathogeneticrole for uric acid in hypertension and cardiovascular and renaldiseaserdquo Hypertension vol 41 no 6 pp 1183ndash1190 2003

[2] V Vitart I Rudan C Hayward et al ldquoSLC2A9 is a newly iden-tified urate transporter influencing serum urate concentrationurate excretion and goutrdquo Nature Genetics vol 40 no 4 pp437ndash442 2008

[3] D I Feig D-H Kang T Nakagawa M Mazzali and R JJohnson ldquoUric acid and hypertensionrdquo Current HypertensionReports vol 8 no 2 pp 111ndash115 2006

[4] S Watanabe D-H Kang L L Feng et al ldquoUric acid hominoidevolution and the pathogenesis of salt-sensitivityrdquo Hyperten-sion vol 40 no 3 pp 355ndash360 2002

[5] M H Alderman ldquoUric acid and cardiovascular riskrdquo CurrentOpinion in Pharmacology vol 2 no 2 pp 126ndash130 2002

[6] C Zoccali R Maio F Mallamaci G Sesti and F PerticoneldquoUric acid and endothelial dysfunction in essential hyperten-sionrdquo Journal of theAmerican Society of Nephrology vol 17 no 5pp 1466ndash1471 2006

[7] E Agabiti-Rosei and G Grassi ldquoBeyond gout uric acid andcardiovascular diseasesrdquoCurrentMedical Research andOpinionvol 29 no 3 pp 33ndash39 2013

[8] S D Anker W Doehner M Rauchhaus et al ldquoUric acid andsurvival in chronic heart failure validation and application inmetabolic functional and hemodynamic stagingrdquo Circulationvol 107 no 15 pp 1991ndash1997 2003

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 BioMed Research International

[9] U M Khosla S Zharikov J L Finch et al ldquoHyperuricemiainduces endothelial dysfunctionrdquo Kidney International vol 67no 5 pp 1739ndash1742 2005

[10] W Doehner N Schoene M Rauchhaus et al ldquoEffects of xan-thine oxidase inhibition with allopurinol on endothelial func-tion and peripheral blood flow in hyperuricemic patients withchronic heart failure results from 2 placebo-controlled studiesrdquoCirculation vol 105 no 22 pp 2619ndash2624 2002

[11] D I Feig and R J Johnson ldquoHyperuricemia in childhoodprimary hypertensionrdquoHypertension vol 42 no 3 pp 247ndash2522003

[12] D I Feig and R J Johnson ldquoThe role of uric acid in pediatrichypertensionrdquo Journal of Renal Nutrition vol 17 no 1 pp 79ndash83 2007

[13] D I Feig D-H Kang and R J Johnson ldquoUric acid andcardiovascular riskrdquoThe New England Journal of Medicine vol359 no 17 pp 1811ndash1821 2008

[14] Q Hong K Qi Z Feng et al ldquoHyperuricemia induces endo-thelial dysfunction via mitochondrial Na+Ca2+ exchanger-mediated mitochondrial calcium overloadrdquo Cell Calcium vol51 no 5 pp 402ndash410 2012

[15] D I Jalal D MMaahs P Hovind and T Nakagawa ldquoUric acidas a mediator of diabetic nephropathyrdquo Seminars in Nephrologyvol 31 no 5 pp 459ndash465 2011

[16] E Manzato ldquoUric acid an old actor for a new rolerdquo Internal andEmergency Medicine vol 2 no 1 pp 1ndash2 2007

[17] K L RockHKataoka and J-J Lai ldquoUric acid as a danger signalin gout and its comorbiditiesrdquo Nature Reviews Rheumatologyvol 9 no 1 pp 13ndash23 2013

[18] T Lyngdoh PMarques-Vidal F Paccaud et al ldquoElevated serumuric acid is associated with high circulating inflammatorycytokines in the population-based colaus studyrdquo PLoSONE vol6 no 5 article e19901 2011

[19] A Bierhaus P M Humpert M Morcos et al ldquoUnderstandingRAGE the receptor for advanced glycation end productsrdquoJournal of Molecular Medicine vol 83 no 11 pp 876ndash886 2005

[20] AM Schmidt S D Yan J-LWautier andD Stern ldquoActivationof receptor for advanced glycation end productsmdashamechanismfor chronic vascular dysfunction in diabetic vasculopathy andatherosclerosisrdquo Circulation Research vol 84 no 5 pp 489ndash497 1999

[21] R Ross ldquoAtherosclerosismdashan inflammatory diseasemdashreplyrdquoThe New England Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[22] A Goldin J A Beckman A M Schmidt and M A CreagerldquoAdvanced glycation end products sparking the developmentof diabetic vascular injuryrdquo Circulation vol 114 no 6 pp 597ndash605 2006

[23] A Soro-Paavonen A M D Watson J Li et al ldquoReceptor foradvanced glycation end products (RAGE) deficiency attenuatesthe development of atherosclerosis in diabetesrdquo Diabetes vol57 no 9 pp 2461ndash2469 2008

[24] S F Yan R Ramasamy and A M Schmidt ldquoThe RAGE axisa fundamental mechanism signaling danger to the vulnerablevasculaturerdquo Circulation Research vol 106 no 5 pp 842ndash8532010

[25] G Basta G Lazzerini M Massaro et al ldquoAdvanced glyca-tion end products activate endothelium through signal-trans-duction receptor RAGE a mechanism for amplification ofinflammatory responsesrdquo Circulation vol 105 no 7 pp 816ndash822 2002

[26] R Ramasamy S F Yan and A M Schmidt ldquoThe rage axisand endothelial dysfunction maladaptive roles in the diabeticvasculature and beyondrdquo Trends in Cardiovascular Medicinevol 15 no 7 pp 237ndash243 2005

[27] U G Andersson andK J Tracey ldquoHMGB1 a pro-inflammatorycytokine of clinical interest introductionrdquo Journal of InternalMedicine vol 255 no 3 pp 318ndash319 2004

[28] H C Wang O Bloom M H Zhang et al ldquoHMG-1 as a latemediator of endotoxin lethality in micerdquo Science vol 285 no5425 pp 248ndash251 1999

[29] W Lee S-K Ku J W Bae and J-S Bae ldquoInhibitory effectsof lycopene on HMGB1-mediated pro-inflammatory responsesin both cellular and animal modelsrdquo Food and ChemicalToxicology vol 50 no 6 pp 1826ndash1833 2012

[30] M M Rabadi M-C Kuo T Ghaly et al ldquoInteraction betweenuric acid and HMGB1 translocation and release from endothe-lial cellsrdquo American Journal of PhysiologymdashRenal Physiologyvol 302 no 6 pp F730ndashF741 2012

[31] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[32] M A Becker and M Jolly ldquoHyperuricemia and associateddiseasesrdquo Rheumatic Disease Clinics of North America vol 32no 2 pp 275ndash293 2006

[33] Y S Kim J P Guevara K M Kim H K Choi D F Heitjanand D A Albert ldquoHyperuricemia and coronary heart disease asystematic review andmeta-analysisrdquoArthritis CareampResearchvol 62 no 2 pp 170ndash180 2010

[34] M Li W Hou X Zhang L Hu and Z Tang ldquoHyperuricemiaand risk of stroke a systematic review and meta-analysis ofprospective studiesrdquoAtherosclerosis vol 232 no 2 pp 265ndash2702014

[35] P O Bonetti L O Lerman and A Lerman ldquoEndothelialdysfunctionmdasha marker of atherosclerotic riskrdquo ArteriosclerosisThrombosis and Vascular Biology vol 23 no 2 pp 168ndash1752003

[36] J Davignon and P Ganz ldquoRole of endothelial dysfunction inatherosclerosisrdquo Circulation vol 109 no 23 supplement 1 ppIII27ndashIII32 2004

[37] L G Sanchez-Lozada M A Lanaspa M Cristobal-Garcıa etal ldquoUric acid-induced endothelial dysfunction is associatedwith mitochondrial alterations and decreased intracellular ATPconcentrationsrdquo NephronmdashExperimental Nephrology vol 121no 3-4 pp e71ndashe78 2013

[38] D B Corry and M L Tuck ldquoUric acid and the vasculaturerdquoCurrent Hypertension Reports vol 8 no 2 pp 116ndash119 2006

[39] P Puddu G M Puddu E Cravero L Vizioli and A MuscarildquoThe relationships among hyperuricemia endothelial dysfunc-tion and cardiovascular diseases molecular mechanisms andclinical implicationsrdquo Journal of Cardiology vol 59 no 3 pp235ndash242 2012

[40] E Biros C S Moran P E Norman et al ldquoAssociation betweenthe advanced glycosylation end product-specific receptor geneand cardiovascular death in older menrdquo PLoS ONE vol 10 no7 Article ID e0134475 2015

[41] M B Manigrasso J Juranek R Ramasamy and AM SchmidtldquoUnlocking the biology of RAGE in diabetic microvascularcomplicationsrdquo Trends in Endocrinology amp Metabolism vol 25no 1 pp 15ndash22 2014

[42] L Lin S Park and E G Lakatta ldquoRAGE signaling in inflam-mation and arterial agingrdquo Frontiers in Bioscience vol 14 no 4pp 1403ndash1413 2009

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 11

[43] P Huebener C Hernandez and R F Schwabe ldquoHMGB1 andinjury amplificationrdquo Oncotarget vol 6 no 27 pp 23048ndash23049 2015

[44] D Tang R Kang H J Zeh and M T Lotze ldquoHigh-mobilitygroup box 1 oxidative stress and diseaserdquo Antioxidants andRedox Signaling vol 14 no 7 pp 1315ndash1335 2011

[45] D J Weber A S Gracon M S Ripsch et al ldquoThe HMGB1-RAGE axis mediates traumatic brain injury-induced pul-monary dysfunction in lung transplantationrdquo Science Transla-tional Medicine vol 6 no 252 pp 5140ndash5144 2014

[46] E Harja D-X Bu B I Hudson et al ldquoVascular and inflamma-tory stresses mediate atherosclerosis via RAGE and its ligandsin apoE-- micerdquo The Journal of Clinical Investigation vol 118no 1 pp 183ndash194 2008

[47] Z-G Luan H Zhang P-T Yang X-C Ma C Zhang and R-XGuo ldquoHMGB1 activates nuclear factor-120581B signaling byRAGEand increases the production of TNF-120572 in humanumbilical veinendothelial cellsrdquo Immunobiology vol 215 no 12 pp 956ndash9622010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom