Anthocyanin Prevents CD40-Activated ProinflammatorySignaling in Endothelial Cells by Regulating

Cholesterol DistributionMin Xia, Wenhua Ling, Huilian Zhu, Qing Wang, Jing Ma, Mengjun Hou, Zhihong Tang,

Lan Li, Qinyuan Ye

Objective—Intracellular tumor necrosis factor receptor-associated factors (TRAFs) translocation to lipid rafts is a keyelement in CD40-induced signaling. The purpose of this study was to investigate the influence of anthocyanin onCD40-mediated proinflammatory events in human endothelial cells and the underlying possible molecular mechanism.

Methods and Results—Treatment of endothelial cells with anthocyanin prevented from CD40-induced proinflammatorystatus, measured by production of IL-6, IL-8, and monocyte chemoattractant protein-1 through inhibiting CD40-inducednuclear factor-�B (NF-�B) activation. TRAF-2 played pivotal role in CD40–NF-�B pathway as TRAF-2 smallinterference RNA (siRNA) diminished CD40-induced NF-�B activation and inflammation. TRAF-2 overexpressionincreased CD40-mediated NF-�B activation. Moreover, TRAF-2 almost totally recruited to lipid rafts after stimulationby CD40 ligand and depletion of cholesterol diminished CD40-mediated NF-�B activation. Exposure to anthocyanin notonly interrupted TRAF-2 recruitment to lipid rafts but also decreased cholesterol content in Triton X-100 insoluble lipidrafts. However, anthocyanin did not influence the interaction between CD40 ligand and CD40 receptor.

Conclusions—Our findings suggest that anthocyanin protects from CD40-induced proinflammatory signaling bypreventing TRAF-2 translocation to lipid rafts through regulation of cholesterol distribution, which thereby mayrepresent a mechanism that would explain the anti-inflammatory response of anthocyanin. (Arterioscler Thromb VascBiol. 2007;27:519-524.)

Key Words: arteriosclerosis � cholesterol � endothelium � inflammation

Atherosclerosis possesses many features of chronic in-flammation and is considered as an immuno-

inflammatory disease.1 Several lines of evidence have dem-onstrated that the immune mediator CD40 and its counterpartCD40 ligand (CD40L) are potent activators in the initiationand development of this disease.2–4

See page 450CD40 is a member of tumor necrosis factor (TNF) receptor

superfamily that provides activation signals not only inantigen-presenting cells5–6 but also in a variety of nonim-mune cells, including vascular cells like endothelial cells(ECs).7 Several studies have indicated that the cytoplasmictail of CD40 lacks intrinsic catalytic activity and signalslargely through its ability to recruit TNF receptor-associatedfactors (TRAFs), adapter proteins that bridge receptors of theTNF family to downstream signaling pathways. Of the 6known mammalian TRAFs, TRAF-2 directly bind to amembrane-distal CD40 cytoplasmic domain and is thought tooccupy a pivotal position in the signaling pathways initiatedby CD40.8–11 Recent reports have described that CD40L

stimulation of B cells renders TRAF-2 largely translocated indetergent-insoluble membrane microdomains or rafts, en-riched in sphingolipids, cholesterol, and glycosyl-phosphatidylinositol-linked proteins.12,13 However, whethersignals induced by CD40 also depends on these specializedmembrane microdomains in vascular cells especially in ECs,which is related to atherogenesis, has not been analyzed indetail.

Anthocyanin is a large family of naturally occurringcompounds and belongs to phytochemicals. Many studieshave shown that it not only imparts color to plants but alsoexhibits pharmacological properties.14,15 Our recent study hasdemonstrated that anthocyanin promotes cholesterol effluxfrom macrophage-derived foam cells, suggesting that antho-cyanin may possess potential function in regulating choles-terol distribution in cells.16 This let us speculate that antho-cyanin may interfere with the recruitment of TRAF-2 in lipidrafts by changing cholesterol distribution and thus interruptsCD40-induced proinflammatory signaling. To confirm thisspeculation, we chose ECs, which are involved in leukocyteextravasation underlying inflammation, to evaluate the effect

Original received August 28, 2006; final version accepted November 19, 2006.From the Department of Nutrition, School of Public Health, Sun Yat-Sen University (Northern Campus), Guangzhou, PR China.Correspondence to Wenhua Ling, MD, PhD, Professor, Dean, Department of Nutrition, School of Public Health, Sun Yat-Sen University (Northern

Campus), Guangzhou, Guangdong Province, PR China 510080. E-mail lingwh@mail.sysu.edu.cn© 2007 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000254672.04573.2d

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of anthocyanin on CD40-induced proinflammatory signalingpathway and uncover its relationship with lipid rafts.

Materials and MethodsAn expanded Materials and Methods section is available in theonline data supplement at http://atvb.ahajournals.org.

Cell CultureHuman umbilical vein endothelial cells were isolated and used in thisstudy.

Immunoprecipitation and ImmunoblottingCellular lysates were immunoprecipitated and the protein complexeswere washed and analyzed by immunoblotting using indicatedantibody.

Luciferase AssayAfter lysis, cellular extracts were assayed for luciferase activityusing luciferase assay system.

Statistical AnalysisData were analyzed statistically ANOVA followed by post-hocstatistical tests.

ResultsAnthocyanin Prevents CD40-MediatedProinflammatory Response in Human ECsTo examine the effect of anthocyanin on CD40-induced endo-thelial cell activation, ECs were stimulated with CD40 cognateligand-sCD40L (5 �g/mL) alone or in the presence of anthocy-anin Cy-3-g or Pn-3-g for 24 hours. The inflammatory responsewas determined by measuring production of proinflammatorycytokines. Exposure to Cy-3-g or Pn-3-g dose-dependentlyinhibited CD40-induced endothelial release of IL-6 (supplemen-tal Figure Ia, available online at http://atvb.ahajournals.org), IL-8(supplemental Figure Ib), and MCP-1 (supplemental Figure Ic).The inhibitory effect of anthocyanin (100 �mol/L) in this modelwas essentially identical to that achieved by neutralizing anti-CD40 antibody. All these data evidenced that anthocyanintreatment prevented CD40-induced inflammation of ECs byrecombinant CD40L.

Anthocyanin Inhibits CD40-Stimulated NF-�BActivation at the Upstream of I�B-�Phosphorylation and Blocks the Acquisition of aProinflammatory EC PhenotypeWe next investigated the effect of anthocyanin on NF-�Bactivation by CD40, which is an important nuclear mediator inimmune responses. Compared with control plasmid, transfectionof ECs with human CD40 resulted in potent and significantinduction of NF-�B reporter activity, which was dose-dependently inhibited by Cy-3-g and Pn-3-g (Figure 1A).

To avoid the limitations of transient transfection systems,we examined the function of anthocyanin in ECs activated bysCD40L. Cy-3-g and Pn-3-g treatment also reduced sCD40L-induced nuclear factor-�B (NF-�B) DNA binding activity ina concentration-dependent manner (Figure 1B). The inhibi-tion of NF-�B activation may, because of the interference byanthocyanin with the upstream level of I�B-� as anthocyanin,also concentration-dependently inhibited CD40-induced

I�B-� activation (supplemental Figure IIa) and promotedI�B-� phosphorylation (supplemental Figure IIb).

To confirm the role of decreased NF-�B activity in theinhibitory function of anthocyanin on EC activation, we usedNF-�B–specific decoy ODN and NF-�B pharmacologicalblocker pyrrolidine dithiocarbamate. Pretreatment of ECs withNF-�B decoy ODN (10 �mol/L) or pyrrolidine dithiocarbamate(10 �mol/L) led to the marked and significant reduction ofCD40-induced cytokines secretion (data not shown).

These data indicated that reduction of CD40-mediatedinflammation by anthocyanin may attribute to the inhibitionof NF-�B activation, which led to ECs acquired a proinflam-matory phenotype.

TRAF-2 Is Essential for CD40-InducedProinflammatory Signaling in ECsPrevious studies suggested that the intracellular signalingmediated by CD40 is regulated by a family of cytoplasmicadaptor molecules TRAFs, which connected TNF receptor todownstream signaling pathways.17–19 We focused our effortson TRAF-2 to explore its role in CD40-mediated signaling.

Figure 1. Anthocyanin inhibits CD40-induced NF-�B activationin ECs. A, ECs were transfected with a control pCMV plasmid(control) or a human CD40 expression plasmid plus NF-�Breporter in the presence of Cy-3-g or Pn-3-g (from 1 to100 �mol/L), then the luciferase activity was determined using�-galactosidase (�-gal) as control. Results of 3 independentexperiments are expressed in RLU. *P�0.05, **P�0.01, or***P�0.001 compared with human CD40. B, ECs were incu-bated with normal media (control) or activated by sCD40L (5�g/mL) alone or in the presence of Cy-3-g and Pn-3-g (from 1to 100 �mol/L) for 1 hour and NF-�B DNA binding activity wasanalyzed by enzyme-linked immunosorbent assay-basedmethod. Similar results obtained in 3 independent experimentsare expressed fold of control. **P�0.01 or ***P�0.001 com-pared with sCD40L-stimulated ECs.

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We pretreated ECs with the specific siRNA against humanTRAF-2 to investigate the effect of TRAF-2 depletion onCD40 signaling. TRAF-2 siRNA significantly decreasedCD40-induced IL-6 (35.47% � 12.38% reduction), IL-8(30.79% � 8.63% reduction), and MCP-1 (39.80% � 4.82%reduction). The TRAF-2 siRNA did not influence PMA-mediated cytokine production in ECs (in which there is noevidence for TRAF-2 involvement, data not shown), demon-strating the specificity of siRNA to TRAF-2–mediated path-way in cytokine production. Compared with control siRNA,TRAF-2 siRNA also significantly reduced CD40-inducedNF-�B reporter (Figure 2A) and sCD40L-activated NF-�Btranscriptional activity (Figure 2B).

To further investigate the casual role of TRAF-2 inmodulation of CD40 signaling, we overexpressed TRAF-2 inECs by transfecting human TRAF-2 construct. Overexpres-sion of TRAF-2 potently enhanced CD40-mediated NF-�Bactivation (supplemental Figure III). However, expression ofdominant negative TRAF-2, which lacks zinc fingers butcontains the C-terminal region essential for binding toCD40,18 almost completely diminished CD40-mediated

NF-�B DNA binding activity (supplemental Figure III),implying the critical role for TRAF-2 in CD40-mediatedsignal transduction. Collectively, all these data demonstratedthat TRAF-2 is an essential and sufficient mediator of theCD40-activated inflammatory events in ECs.

Recruitment of TRAF-2 to Lipid Rafts DuringCD40 SignalingAlthough TRAF-2 is critical to CD40 signaling, how TRAF-2delivers the CD40-mediated signal to downstream is lessunderstood An emerging evidence in cell surface receptorsignaling is detergent-resistant liquid-ordered lipid membranemicrodomains, or lipid rafts.20,21

To demonstrate the engagement of TRAF-2 in CD40 signal-ing, we used 3 separate approaches. First, we used immunopre-cipitation to detect TRAF-2 interaction with CD40. In theunstimulated state, CD40 immunoprecipitates were not detectedby TRAF-2 antibody (lane 1). CD40L stimulation resulted inobvious detection of TRAF-2 in CD40 immunoprecipitates (lane2). Control experiments validated the nature of the TRAF-2 andCD40 immunoprecipitates (lane 3 and lane 4; supplementalFigure IV).

As a second approach, ECs were stimulated with sCD40L andthe Triton X-100 insoluble/soluble pellets were isolated. Asdemonstrated in Figure 3A, all of TRAF-2 from unstimulatedECs (0 minutes) appeared in the detergent-soluble fraction. Asignificant translocation of TRAF-2 from soluble to insolublefractions was already observed as early as 5 minutes aftersCD40L addition and totally recruited to insoluble fraction after10 minutes of stimulation, thereafter they progressively declinedafter 30 minutes.

Figure 2. TRAF-2 is essential in CD40-induced signaling in ECs.A, Human CD40 expression plasmid plus NF-�B reporter withTRAF-2 siRNA were simultaneous transfected into cells with�-gal reporter as internal reference. Cellular extracts wereassayed for NF-�B luciferase activity. Results of 3 independentexperiments are expressed as relative light units (RLU).***P�0.001 compared with human CD40. B, ECs were trans-fected with either control siRNA or TRAF-2 siRNA, then sCD40L(5 �g/mL) was added for 1 hour and NF-�B transcriptionalactivity was measured. Results obtained in 3 independentexperiments are expressed as fold of control. ***P�0.001 com-pared with sCD40L-stimulated ECs.

Figure 3. TRAF-2 recruitment to lipid rafts by activation ofsCD40L. A, ECs were stimulated with sCD40L for the indicatedtime points, and Triton X-100 soluble (S) and insoluble lipid rafts (R)fractions were isolated. Equal aliquots of the fractions were sub-jected to SDS-PAGE, and the protein distribution was assessed byimmunoblotting using specific anti-TRAF-2 antibody. B, ECs werestimulated with flag-tagged sCD40L for the indicated time points,and Triton X-100 soluble (S) and insoluble lipid raft (R) fractionswere isolated. Engaged signaling complexes were immunoprecipi-tated using anti-Flag antibody. Immunoprecipitates and corre-sponding total cell lysates were subjected to SDS-PAGE and im-munoblotted using specific antibody.

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Next, we investigated the CD40–TRAF-2 signal transductionpathways by means of analysis of their signaling complexes.Additionally, we studied whether the signaling complex elicitedby the engagement of endogenous CD40 occurred within oroutside of microdomains. To this end, ECs were stimulated withFlag-tagged CD40L, lipid rafts were isolated, and the engagedCD40-TRAF-2 signaling complex were analyzed by immuno-precipitation using an anti-Flag antibody. For comparison, thetotal amount of receptor was immunoprecipitated by adding theligand and anti-Flag antibody to the soluble and raft fractions ofunstimulated cells. Before stimulation (0 minutes), TRAF-2 andCD40 complex was not formed and could not be detected inlipid rafts. On CD40L stimulation, the TRAF-2 and CD40signaling complex was immediately formed and then completelytranslocated to lipid rafts in 5 minutes. The TRAF-2 complexwas completely recruited to lipid rafts after 10 minutes (Figure3B). Taken together, these observations provide strong evidencefor the essential role of TRAF-2 recruitment to lipid rafts inCD40-mediated signal transduction.

Interference With Lipid Rafts CompositionInduces the Switch of CD40 Signaling fromNF-�B ActivationCholesterol has been postulated to be a crucial structural com-ponent of rafts.22 To demonstrate directly the involvement oflipid rafts in CD40 signaling, we used �- MCD,23 which hasbeen shown to be a useful tool in extracting cholesterol frombiological membranes and disrupted membrane microdomains,to examine the role of lipid rafts in CD40-mediated NF-�Bactivation. Disruption of lipid rafts by �-MCD almost com-pletely abrogated TRAF-2 engagement with CD40 (supplemen-tal Figure Va, lane 2). This effect is not attributable to the loss ofCD40 from this fraction with �-MCD treatment alone (supple-mental Figure Va, lane 3). Compared with untreated ECs (Figure

4, top), �-MCD treatment also totally interrupted TRAF-2translocation to lipid lipids after sCD40L stimulation (Figure 4,middle). However, �-MCD, which is an inactive analogue of�-MCD and unable to deplete cholesterol, was shown to exertno effect on CD40-induced TRAF-2 translocation (Figure 4,bottom). We next analyzed the effect of MCD on CD40-inducedNF-�B activation by monitoring the phosphorylation of I�B-�.NF-�B activation was observed 5 minutes after stimulation ofECs with sCD40L, which was totally abolished by �-MCD(supplemental Figure Vb, top). However, �-MCD did not exertany impact on CD40-induced NF-�B activation (supplementalFigure Vb, bottom). Collectively, these results suggested thatinterference with lipid rafts organization abolished TRAF-2recruitment to this microdomain and switched its mediatedsignaling.

Anthocyanin Blocks TRAF-2 Recruitment to LipidRafts Via Regulating Cholesterol Redistribution ofMembrane Lipid RaftsNext, the effect of anthocyanin on TRAF-2 recruitment tolipid rafts was examined. Notably, we found that anthocyaninCy-3-g and Pn-3-g pretreatment before sCD40L stimulationdose-dependently decreased TRAF-2 recruitment in lipidrafts and increased TRAF-2 engagement in soluble fractions,indicating that anthocyanin blocked CD40- induced translo-cation of TRAF-2 to lipid rafts (Figure 5A).

Figure 4. �-MCD induces the switch of CD40-induced signaling.ECs were left untreated, or pretreated with �-MCD (20 mmol/L)or �-MCD (20 mmol/L) for 30 minutes, then stimulated withsCD40L for the indicated time points, and lysed in 0.5% Brij 78for 1 hour, followed by immunoprecipitation using the anti-CD40antibody and the engaged TRAF-2 was analyzed by immuno-blotting using anti-TRAF-2 antibody.

Figure 5. Anthocyanin prevents TRAF-2 recruitment to lipidrafts and regulates cholesterol distribution in ECs. A, ECswere pre-exposed to anthocyanin Cy-3-g and Pn-3-g at theconcentrations of 1, 10, and 100 �mol/L for 12 hours andthen stimulated by 5 �g/mL sCD40L for 10 minutes. Afterthat, the Triton X-100 soluble and insoluble lipid rafts frac-tions were isolated and the protein distribution was assessedby immunoblotting. B, ECs were pretreated with anthocyaninfor 12 hours, then stimulated with 5 �g/mL sCD40L for 6hours, the distribution of total cholesterol in insoluble lipidrafts fractions was measured by enzymatic method. *P�0.05,**P�0.01, or ***P�0.001 compared with control and sCD40L.

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Having demonstrated that anthocyanin prevented CD40L-induced TRAF-2 recruitment in lipid rafts, we attempted tocharacterize the underlying possible molecular mechanism. Tothis point, we first designed to analyze whether anthocyanin mayinterfere with CD40L binding to CD40 receptor using flowcytometry. In unstimulated ECs, no detectable CD40L bindingcould be found, which was significantly augmented by stimula-tion of fluorescein isothiocyanate–CD40L. The interaction ofCD40L with CD40 receptor was not influenced after addition ofanthocyanin from 1 �mol/L to 100 �mol/L, excluding thepossibility that anthocyanin inhibited CD40 signaling by inter-rupting ligand binding to receptor (supplemental Figure VI).

We further investigated the perturbing effect of anthocyaninon the membrane lipid rafts structure by determining thedistribution of cholesterol in lipid rafts and soluble fractions.Anthocyanin slightly reduced cholesterol content from the sol-uble fraction (supplemental Figure VII), but efficiently anddose-dependently depleted cholesterol from the Triton X-100insoluble lipid rafts (Figure 5B).

DiscussionThe major findings of our current study demonstrate thatanthocyanin, namely, Cy-3-g and Pn-3-g, disrupts TRAF-2recruitment to membrane lipid rafts by reducing cholesteroldistribution in lipid rafts of membrane in human ECs. Thedepletion of TRAF-2 translocation by anthocyanin in the endo-thelial monolayer limits CD40-mediated activation of NF-�B,thus leading to the reduction of proinflammatory activation inECs responsible to CD40 stimulation (Figure 6). CD40 signalingis a major trigger eliciting a proinflammatory reaction andpromoting formation of atherosclerotic lesions.24–26 Inhibition ofCD40-mediated inflammatory response by anthocyanin maythereby partially explain its anti-atherogenetic effects. To ourknowledge, these results are the first demonstration of anthocy-

anin on the anti-inflammatory mode with respect to CD40signaling in ECs. The data also further knowledge of intracel-lular signaling pathway that is activated by CD40 on ECs,particularly the role of lipid rafts.

Today, multiple lines of evidence suggested that interaction ofCD40 and its counterpart CD40L plays a crucial role in the onsetand maintenance of inflammatory response, which is implicatedin atherogenesis.27–30 Present study clearly shows that exposureof ECs to CD40 physiological ligand CD40L promotes func-tional cellular CD40 inflammatory signaling by increasing therelease of IL-6, IL-8, and MCP-1. This effect is not attributableto the cytotoxicity of anthocyanin because the dosage of antho-cyanin used in this experiment did not affect the cell viability orcause significant cytotoxicity on ECs which was determined byMTT cell proliferation assay, lactate dehydrogenase releaseassay, and trypan exclusion test (data not shown). These proin-flammatory molecules then may attract direct T lymphocytesand macrophages to the atheroma and maintain chronic inflam-mation, promoting the initiation and progression of atheroscle-rotic lesion. It has been reported that CD40 signaling in ECs maybe involved with activation of transcription mediators such asNF-�B.7 In consistence with this concept, we find that transfec-tion of CD40 plasmid or stimulation with sCD40L in ECssignificantly increases NF-�B reporter and DNA binding activ-ity. Preexposure of ECs with NF-�B decoy ODN or pyrrolidinedithiocarbamate interrupts CD40-induced cytokines production,indicating that NF-�B activation is essential in CD40-mediatedinflammation. NF-�B belongs to a major family of transcrip-tional mediators, which are located in the intracellular nucleus.Because CD40 is a membrane receptor, question arises how theextracellular signals are tranduced from the cell surface to thenucleus activation.

Recent studies have demonstrated that TRAFs, including 6TRAF proteins, associate with and transduce signals from TNFreceptor family members, may act as an important adapter ofproinflammatory signaling induced by CD40.17–19,31 TRAF-2appears to interfere with CD40 signaling through activatingNF-�B DNA binding activity as TRAF-2 siRNA significantlyreduced induction of NF-�B activation and cytokines release byCD40. Overexpression of TRAF-2 in ECs leads to increasedNF-�B activation induced by CD40. Although the functions ofTRAF proteins remain enigmatic, recent reports demonstratedthat TRAF may associate with lipid rafts and then transducesignals from TNF receptor family members.12,17,21 But untilnow, the functional importance of lipid rafts in CD40-mediatedsignal transduction especially in vascular cells is still poorlyunderstood. Remarkably, in this study, we found that sCD40Lstimulation of ECs renders the rapid and dramatic recruitment ofTRAF-2 bind to CD40 in the detergent-insoluble membranemicrodomains or rafts. The microdomain integrity is essentialfor CD40-mediated NF-�B activation since �-MCD–mediatedcholesterol depletion was shown to block the translocation ofTRAF-2 molecule to lipid rafts and hampered their associationwith CD40. �-MCD, which is unable to deplete cholesterol,exerts no effect on CD40-induced TRAF-2 translocation. Takentogether, these findings suggest that CD40-mediated TRAF-2interaction with lipid rafts components is critical for CD40-induced downstream proinflammatory events in ECs. However,the mechanism why the alteration of TRAF-2 translocation to

Figure 6. The schematic summary to demonstrate the molecularmechanism of anthocyanin on CD40-induced inflammatory sig-naling through regulating cholesterol distribution. Anthocyanintreatment decreases cholesterol content in Triton X-100 insolu-ble lipid rafts resulting in diminished formation of CD40–TRAF-2complex in lipid rafts. This disrupts CD40-induced NF-�B acti-vation, prevents proinflammatory events such as IL-6, IL-8, andMCP-1 production induced by CD40, and limits inflammation.

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lipid rafts affects CD40-mediated signaling remains unclear andneeds further elucidation.

Anthocyanins are the most important plant pigments. Althoughin recent years, emerging reports have been evidenced the impor-tance of anthocyanins as dietary antioxidants for prevention ofoxidative damage, several studies gradually focused on the itsanti-inflammatory effect.32,33 In the current study, we show thatanthocyanin prevents CD40-induced inflammatory response viadecreasing CD40-mediated NF-�B activation. Anthocyanintreatment also inhibits CD40-induced translocation of TRAF-2to lipid rafts in a dose-dependent fashion and this effect is notrelated to the impact of anthocyanin on CD40L binding to CD40receptor. We then analyzed the effect of anthocyanin on thestructure and composition of lipid rafts which is crucial for lipidrafts. More strikingly, we find that anthocyanin exposure signif-icantly depletes cholesterol from the Triton X-100 insolublelipid rafts, indicating strongly that anthocyanin blocks thetranslocation of TRAF-2 to lipid rafts in the cellular membraneand thus interrupts CD40-induced signaling.

In summary, we demonstrate that anthocyanin reduced cho-lesterol content in membrane insoluble lipid rafts in ECs,resulting in the decrease of TRAF-2 translocation to lipidrecruitment and inhibition of CD40-induced inflammatory sig-naling pathway in ECs. These findings suggest that anthocyaninmay act as a lipid-dependent regulator of inflammatory responseand provide a novel insight into the therapeutic implications ofanthocyanin in many chronic inflammatory-related diseases.

Sources of FundingThis work was supported by the research grants from NationalNatural Science Foundation of China Research grants 30025037,30371215, 30571568, China Medical Board of New York Inc (grantCMB 98-677), the National Basic Research Program (973 Program,No. 2006CB503902), and the Team Project of the Science Founda-tion of Guangdong Province.

DisclosuresNone.

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29. Zhang-Hoover J, Sutton A, Stein-Streilein J. CD40/CD40 ligand inter-actions are critical for elicitation of autoimmune-mediated fibrosis in thelung. J Immunol. 2001;166:2556–2563.

30. Schonbeck U, Libby P. CD40 signaling and plaque instability. Circ Res.2001;89:1092–1103.

31. Davies CC, Mak TW, Young LS, Eliopoulos AG. TRAF6 is required forTRAF2- dependent CD40 signal transduction in nonhemopoietic cells.Mol Cell Biol. 2005;25:9806–9819.

32. Kalin R, Righi A, Del Rosso A, Bagchi D, Generini S, Cerinic MM, Das DK.Activin, a grape seed-derived proanthocyanidin extract, reduces plasmalevels of oxidative stress and adhesion molecules (ICAM-1, VCAM-1 andE-selectin) in systemic sclerosis. Free Radic Res. 2002;36:819–825.

33. Kim HJ, Tsoy I, Park JM, Chung JI, Shin SC, Chang KC. Anthocyaninsfrom soybean seed coat inhibit the expression of TNF-�-induced genesassociated with ischemia/reperfusion in endothelial cell by NF-�B-dependent pathway and reduce rat myocardial damages incurred by is-chemia and reperfusion in vivo. FEBS Lett. 2006;580:1391–1397.

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IL-6

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Control sCD40L 1 10 100 1 10 100 anti-CD40L

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c

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MC

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L)

µmol/L Pn-3-g µmol/L Cy-3-g

Figure I. Anthocyanin inhibits CD40-induced inflammatory response in ECs. ECs were stimulated with 5 µg/mL sCD40L alone or in the presence of Cy-3-g or Pn-3-g (from 1 to 100 µmol/L) for 24 hours. Thereafter, the proinflammatory molecules including (a) IL-6, (b) IL-8 and (c) MCP-1 release were measured by ELISA in supernatants of ECs. Data are representative of 3 independent experiments. *P<0.05, **P<0.01 or ***P<0.001 compared to sCD40L-stimulated ECs.

IκB-α reporter

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Control HumanCD40

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Figure II. Anthocyanin inhibits CD40-stimulated IκB-α activation and phosphorylation. a, ECs were transfected with a control pCMV plasmid (control) or a human CD40 expression plasmid plus IκB-α reporter in the presence of Cy-3-g or Pn-3-g (from 1 to 100 µmol/L), then the luciferase activity was determined using β-gal as control. Results of 3 independent experiments are expressed in RLU. *P<0.05, **P<0.01 or ***P<0.001 compared to human CD40. b, ECs were incubated with normal media (Control) or activated by sCD40L (5 µg/mL) alone or in the presence of Cy-3-g and Pn-3-g (from 1 to 100 µmol/L) for 1 hour and IκB-α phosphorylation was analyzed by ELISA based method. Similar results obtained in 3 independent experiments are expressed fold of control. **P<0.01 or ***P<0.001 compared to sCD40L-stimulated ECs.

NF-κB DNA binding activity

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Figure III. DnTRAF-2 reduces CD40-induced NF-κB transcriptional activity in ECs. ECs were transfected with either control (pcDNA3.1) or pcDNA3.1-TRAF-2, then ECs were stimulated with sCD40L (5 µg/mL) and NF-κB transcriptional activity was measured. Results acquired from 3 independent experiments are expressed as fold of control. *P<0.05 or ***P<0.001 compared to TRAF-2+ sCD40L-treated ECs.

lane 1 lane 2 lane 3 lane 4

- + - - sCD40L (5 µg/mL)

IP CD40 CD40 TRAF-2 CD40

IB TRAF-2 TRAF-2 TRAF-2 CD40 Figure IV. TRAF-2 engagement with CD40 in ECs by activation of sCD40L. ECs were either activated with sCD40L or left untreated as indicated. Cell lysates were subjected to immunoprecipitation (IP) and then immunoblotted (IB) with the anti-TRAF-2 or anti-CD40 antibody, respectively.

a

lane 1 lane 2 lane 3 lane 4 lane 5 - + + - - β-MCD

- + - - - sCD40L

IP CD40 CD40 CD40 TRAF-2 CD40

IB TRAF-2 TRAF-2 CD40 TRAF-2 CD40

b

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IκB-α

sCD40L - + - + untreated β-MCD

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IκB-α

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Figure V. Beta-MCD abrogates TRAF-2 engagement with CD40 and interrupts CD-induced NF-κB activation. a, ECs were pretreated or not with β-MCD for 30 minutes, then cells were activated with sCD40L as indicated. Cell lysates were subjected to immunoprecipitation (IP) and then analyzed by immunoblotting (IB) with the indicated antibody. b, ECs were pretreated with β-MCD (20 mmol/L) or α-MCD (20 mmol/L) for 30 minutes, washed and then stimulated with sCD40L as indicated. Cell lysates were subjected to immunblotting and NF-κB activation was monitored by the phosphorylation of IκB-α.

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Figure VI. Anthocyanin does not influence CD40L binding with CD40 receptor in ECs. ECs were preincubated on ice for 1 hour with anthocyanin Cy-3-g or Pn-3-g, followed by the addition of the FITC-labeled CD40L (5 µg/mL) for an additional 30 minutes on ice. Cells were washed and the CD40L binding was detected by flow cytometry.

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Figure VII. Anthocyanin slightly decrease cholesterol content from the soluable fractions in ECs. ECs were pretreated with anthocyanin for 12 hours, then stimulated with 5 µg/mL sCD40L for 6 hours, the distribution of total cholesterol in Triton X-100 soluble fraction was measured by enzymatic method. *P<0.05, **P<0.01 or ***P<0.001 compared to Control and sCD40L.

Li and Qinyuan YeMin Xia, Wenhua Ling, Huilian Zhu, Qing Wang, Jing Ma, Mengjun Hou, Zhihong Tang, Lan

Regulating Cholesterol DistributionAnthocyanin Prevents CD40-Activated Proinflammatory Signaling in Endothelial Cells by

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