Takahide Kohro, Hiroyuki Itabe, Tatsuhiko Kodama and Yukio MaruyamaKeiichi Kawamura, Kazunobu Ishikawa, Youichiro Wada, Satoshi Kimura, Hayato Matsumoto,

DysfunctionBilirubin From Heme Oxygenase-1 Attenuates Vascular Endothelial Activation and

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Bilirubin From Heme Oxygenase-1 Attenuates VascularEndothelial Activation and Dysfunction

Keiichi Kawamura, Kazunobu Ishikawa, Youichiro Wada, Satoshi Kimura, Hayato Matsumoto,Takahide Kohro, Hiroyuki Itabe, Tatsuhiko Kodama, Yukio Maruyama

Objective—Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation, has recently been considered tohave protective roles against various pathophysiological conditions. Since we demonstrated that HO-1 overexpressioninhibits atherosclerotic formation in animal models, we examined the effect of HO modulation on proinflammatorycytokine production, endothelial NO synthase (eNOS) expression, and endothelium-dependent vascular relaxationresponses.

Methods and Results—After HO-1 induction by heme arginate (HA), vascular endothelial cell cultures were exposed tooxidized low-density lipoprotein (oxLDL) or tumor necrosis factor-� (TNF-�). HA pretreatment significantly attenuatedthe production of vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and macrophage colony-stimulating factor, suggesting that HO-1 induction attenuates proinflammatory responses. In addition, HO-1 overex-pression also alleviated endothelial dysfunction as judged by restoration of attenuated eNOS expression after exposureto oxLDL and TNF-�. Importantly, impaired endothelium-dependent vascular relaxation responses in thoracic aorticrings from high-fat–fed LDL receptor knockout mice were also improved. These effects were observed by treatmentwith bilirubin not by carbon monoxide.

Conclusions—These results suggest that the antiatherogenic properties of HO-1 may be mediated predominantly throughthe action of bilirubin by inhibition of vascular endothelial activation and dysfunction in response to proinflammatorystresses. (Arterioscler Thromb Vasc Biol. 2005;25:155-160.)

Key Words: heme oxygenase � oxidized LDL � endothelial nitric oxide synthase � bilirubin � carbon monoxide

Vascular endothelial cell activation by oxidized LDL(oxLDL) and cytokines such as tumor necrosis factor-�

(TNF-�) is considered to play an essential role in thedevelopment of atherosclerotic lesions.1 Activated endotheli-al cells produce adhesion molecules, chemokines, and growthfactors such as vascular cell adhesion molecule-1 (VCAM-1),monocyte chemotactic protein-1 (MCP-1), and macrophagecolony-stimulating factor (MCSF).2–4 Numerous studies haveshown that these molecules promote multiple steps in theformation of atherosclerotic lesion.1,3,4 Endothelial dysfunc-tion, which is associated with decreased bioavailability of NOfrom endothelial NO synthase (eNOS), is also considered toplay an important role in atherogenesis.1,5 NO formed byeNOS has been shown to contribute to vascular smoothmuscle cell relaxation and inhibition of platelet aggregation.1

Heme oxygenase (HO) catalyzes the rate-limiting step ofheme degradation in mammals.6 The products of the reactionare biliverdin, carbon monoxide (CO), and free iron. It hasbeen suggested that biliverdin and CO have cytoprotectiveeffects against various cellular stresses.7–9 We demonstrated

that the inducible form of HO (HO-1) is induced in culturedvascular endothelial cells, smooth muscle cells, and macro-phages by oxidized low-density lipoprotein (oxLDL), andthat high expression of HO-1 results in attenuation of mono-cyte chemotaxis by oxLDL.9 In fact, HO-1 is expressed inatherosclerotic lesions.10,11 We also demonstrated that over-expression of HO-1 inhibits the formation of atheroscleroticlesions by inhibiting lipid peroxidation and by affecting NOmetabolism.11,12 These data suggest that HO-1 as an intrinsicantioxidant plays an important role against atherogenesis.However, it is still unknown1 which proinflammatory mole-cules are involved in the biological action of HO-1 and2

which HO-1 reaction product is predominantly responsiblefor vascular endothelial protection.

In this study, we screened for changes in genes after hemearginate (HA) treatment of vascular endothelium using cDNAmicroarray analyses to eliminate the possibility that use ofheme derivatives to induce HO-1 may result in effects ongenes other than the HO-1 gene. These analyses revealed thatthe HA treatment used in this study had the strongest effect on

Original received February 2, 2004; final version accepted October 3, 2004.From the First Department of Internal Medicine (K.K., K.I., H.M., Y.M.), Fukushima Medical University, the Department of Molecular Biology and

Medicine (Y.W., T. Kohro, T. Kodama), University of Tokyo, and the Department of Biological Chemistry (H.I.), School of Pharmaceutical Sciences,Showa University, Japan.

Correspondence to Kazunobu Ishikawa, MD, PhD, FAHA, First Department of Internal Medicine, Fukushima Medical University, Fukushima, Japan,960-1295. E-mail kishikaw@fmu.ac.jp

© 2005 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000148405.18071.6a

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the HO-1 gene and that effects on other genes were minimal.Endothelial HO-1 overexpression significantly attenuatedproduction of inflammatory mediators and reversed the de-crease in eNOS by oxLDL and TNF-�. In addition, HO-1overexpression also improved the impaired vasodilatory re-sponses of aortic segments treated with oxLDL. Importantly,these effects on vascular endothelium were predominantlyobserved by treatment with bilirubin not by CO. These resultssuggest the possibilities that HO-1 via the action of bilirubinis involved in suppression of endothelial dysfunction andactivation in response to proinflammatory stresses.

MethodsReagentsTNF-� was obtained from R&D systems and HA from HuhtamakiOy Pharmaceuticals. Tricarbonyldichlororuthenium (II) dimer([Ru(CO)3Cl2]2) was from Stream Chemicals. All other reagentswere obtained from Sigma-Aldrich unless indicated otherwise. HA,Sn-protoporphyrin IX (SnPP IX), and bilirubin solutions wereprepared in the dark.

Cell CultureHuman aortic endothelial cells (HAECs) were purchased fromClonetics. In all experiments, cells were used at passage 4-6. HO-1activities were modulated by either 1 to 10 �mol/L HA for 16 hoursor 10 �mol/L SnPP IX for 2 hours as described previously.9 Togenerate CO in cell cultures, tricarbonyldichlororuthenium (II) dimer([Ru(CO)3Cl2]2), a CO-releasing compound,13 was used. The amountof CO released was assessed spectophotometrically by measuring theconversion of deoxymyoglobin to carbonmonoxy myoglobin. Theamount of CO released from 50 �mol/L [Ru(CO)3Cl2]2 in culturemedium was �38.7�2.5 �mol/L in medium (data not shown). Cellspretreated with bilirubin were not exposed to light to preventdegradation.

Microarray Analysis of cRNA andQuantitative AnalysisHAECs were cultured with 5 �mol/L HA and harvested after 16hours. First-strand cDNA was generated with 10 �g of mRNA usingT7-linked oligo(dT) primer (Takara). After second-strand synthesis,in vitro transcription was performed with biotinylated UTP and CTP(Enzo Diagnostics). Resultant cRNA was purified on an affinityresin column (RNeasy; Qiagen). Biotinylated cRNA (40 �g) wasfragmented and hybridized to oligonucleotide gene chip, human FLarray (Affymetrix), and 6416 genes were analyzed as describedpreviously.14 Quantitative analyses were performed with GeneChipSoftware (Affymetrix). The average differences and fold changeswere defined and calculated as described previously.15

Lipoprotein Isolation and ModificationLDL was isolated from the sera of normal blood donors bydensity-gradient ultracentrifugation. OxLDL was prepared byincubating freshly prepared LDL at 37°C for 24 hours in PBS (pH7.4) containing 50 �mol/L CuSO4 and then dialyzing extensivelyagainst PBS.

Western Blot AnalysisCell cultures and mouse aorta were washed, lysed, and homogenizedin 10 mmol/L Tris-HCl (pH 7.4) containing 0.1% sodium dodecylsulfate and a protease inhibitor cocktail (Boehringer Mannheim). Atotal of 20 �g of cell lysate and tissue homogenates was electropho-resed on 10% SDS-PAGE, blotted onto polyvinyliden difluoridemembranes (ATTO), blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, and incubated withpolyclonal anti–HO-1 antibody (StressGen) or monoclonal anti-eNOS antibody (Transduction Laboratories). Bound antibody was

detected using the enhanced chemiluminescence detection system(Amersham Pharmacia). The protein content was determined using aDC protein assay kit (BioRad).

HO-1 AssayHO activity was measured in the microsomal fractions by determin-ing the amount of bilirubin formed as described previously.9

ELISA for Inflammatory MediatorsELISA for VCAM-1, MCP-1, and MCSF was performed with kits(R&D Systems) using conditioned media from HAEC culturesaccording to manufacturer protocol.

RNA Extraction and Northern Blot AnalysisTotal RNA was isolated with TRIzol reagent (GIBCO/BRL). A totalof 20 �g of total RNA was electrophoresed, transferred to a nylonmembrane (MSI), and hybridized with 32P-labeled rat HO-1 andVCAM-1 cDNAs as described previously.9

Plasma Lipids and Lipid Peroxidation AssayBlood was collected from mice fasted overnight. Total plasmacholesterol, triglyceride, and high-density lipoprotein (HDL) con-centrations were determined enzymatically. Plasma lipid hydroper-oxides were measured by the methylene blue hemoglobin method.16

Animal Handling and VascularRelaxation ResponseAll animal experiments were conducted in accordance with theguidelines of the Fukushima Medical University animal researchcommittee. Two- to 3-month-old female LDL-receptor knockoutmice with a C57BL/6J background (C57BL/6J-Ldlrtm1Her) were fedeither standard rodent chow containing 5% fat and 0.075% choles-terol diet (C group; n�6) or high-fat diet containing 15% fat, 1.25%cholesterol, and 0.5% cholic acid (Oriental Bio Service Kanto Inc.;HF group; n�6). The high-fat diet was started 1 week before theexperiment for vascular endothelium response. To overexpressHO-1,11 HA (25 mg/kg) was injected intraperitoneally every otherday during high-fat diet (HF�HA group; n�6). To examine theeffects of CO, CO at 60 ppm was administered in a chamber for 2hours per day during high-fat diet (HF�CO group; n�6). Toexamine the effects of bilirubin, bilirubin (25 mg/kg) was injectedintraperitoneally every other day during high-fat diet (HF�BRgroup; n�6).

To examine endothelium-dependent vascular relaxation responses,isolated thoracic aortic strips were prepared. After euthanization, thedescending thoracic aorta was excised, washed, and cut into aorticrings (2 mm). Aortic rings were then suspended in organ chambersin the Krebs’ buffer saturated with a 95% O2, 5% CO2 gas mixture.After equilibration for 90 minutes, the aorta was contracted with1 �mol/L phenylephrine, and the endothelium-dependent and-independent responses were examined with acetylcholine (10�9 to10�5 mol/L) and sodium nitroprusside (10�9 to 10�5 mol/L).

Data AnalysisAll values are expressed as means�SD. Significant difference wasdetermined by 1-way ANOVA with the Fisher post hoc test. P�0.05was considered significant.

ResultsHO-1 Induction in Vascular Endothelial Cellsby HATo induce HO-1 in cultured vascular endothelial cells, wetreated them with micromolar concentrations of HA. Figure1A shows the dose-dependent induction of HO-1 protein andenzyme activity by HA. We did not observe any apparenttoxic effects of HA as judged by trypan blue exclusion orlactate dehydrogenase release (data not shown).

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To exclude pleiotropic effects of HA on genes other thanHO-1, 6416 genes were screened with the GeneChipSystem before and after treatment with 5 �mol/L HA for16 hours, the condition used to induce HO-1 in this study.The Table shows the genes, which were induced �3-foldor were downregulated �2-fold by HA. HO-1 was the genethat responded most and was induced up to 13-fold,although HA also upregulated several other genes such asNAD(P)H:menadione oxidoreductase and cytochromeP450 (CYP1A2), which have an antioxidant responseelement (ARE).17 In contrast, HA treatment reduced ex-pression levels of 4 genes to �50%. The Table alsoincludes the genes that are thought to be involved inendothelial activation or dysfunction, but HA treatment didnot significantly change their expression. These resultssuggest that HA treatment induces HO-1 in a relativelyspecific manner.

HO-1 Induction Attenuates ProinflammatoryResponses to Oxidized LDL and TNF-� inVascular Endothelial CellsTo determine the effect of HO-1 on proinflammatory re-sponses in vascular endothelium, we first examined theproduction of VCAM-1 in response to TNF-� (2 ng/mL) byELISA (Figure 1B). HA pretreatment (5 �mol/L) itself didnot induce the production and secretion of VCAM-1 into theconditioned media. Interestingly, VCAM-1 production afterTNF-� exposure was significantly reduced by pretreatments

with 1, 5, and 10 �mol/L HA. These effects were abolishedby HO inhibitor SnPP IX (10 �mol/L). In addition, HApretreatment also attenuated induction of VCAM-1 mRNAafter TNF-� exposure (Figure 1C).

To examine the responsible HO-1 product that suppressesVCAM-1 production in response to TNF-�, we pretreatedcells with [Ru(CO)3Cl2]2 (50 �mol/L), a CO-releasing com-pound,13 or bilirubin (5 �mol/L).9 Interestingly, this inhibi-tory effect on VCAM-1 production was predominantly ob-served by treatment with bilirubin not by CO. These resultssuggest a potential role for HO-1 and bilirubin in suppressingproinflammatory interactions between vascular endothelialcells and leukocytes.

To examine the effects of HO-1 on other proinflamma-tory molecules, production of MCP-1 and MCSF in vas-cular endothelium after exposure to oxLDL was alsoexamined. After pretreatment with HA, SnPP IX,[Ru(CO)3Cl2]2, and bilirubin, cells were exposed to oxLDL(50 �g/mL) or TNF-� (2 ng/mL) for 3 hours, washed,incubated in fresh media for another 3 hours, and the levelsof MCP-1 and MCSF were measured in the conditionedmedia by ELISA (Figure I, available online at http://atvb.ahajournals.org). Production of MCP-1 and MCSFwas also significantly suppressed by HA pretreatment.Similar effects of bilirubin on production of these mole-cules were also observed.

Figure 1. HO-1 induction by HA and bilirubin treatmentresults in attenuation of VCAM-1 production caused byTNF-� in HAECs. A, HO-1 protein expression (left panel) andactivities (right panel) after HA treatments. Cells wereexposed to 0, 1, 5, and 10 �mol/L HA for 16 hours and ana-lyzed by Western blotting and HO assay. B, HA and bilirubinpretreatment attenuates VCAM-1 production caused byTNF-�. Cells were pretreated with HA for 16 hours to induceHO-1 or with SnPP IX (10 �mol/L) for 2 hours to inhibit HO-1.To observe the effects of CO and bilirubin, cells were pre-treated with [Ru(CO)3Cl2]2 (50 �mol/L), or bilirubin (5 �mol/L)in the dark for 2 hours, respectively. Then cells were exposedto TNF-� (2 ng/mL) for 12 hours, washed 3�, and incubatedin fresh media for another 12 hours. Media collected between12 and 24 hours after TNF-� exposure were analyzed forVCAM-1 production (ng/mL) by ELISA in duplicate. Datashown are representative for 3 independent experiments.*P�0.001; **P�0.005. C, HA pretreatment attenuatesVCAM-1 mRNA expression caused by TNF-�. Total RNA wasprepared 6 hours after TNF-� exposure.

cDNA Microarray Analysis of Human Vascular Endothelial CellsAfter Heme Arginate Treatment

Accession No. GeneAverage

DifferenceFold

Change

Upregulated

X06985 HO-1 2202.2 13.0

J03934 NAD(P)H:menadione oxidoreductase 326.6 4.5

M31667 Cytochrome P450 (CYP1A2) 99.9 3.8

M80244 E16 199.9 3.3

X12451 Pro-cathepsin L 158.7 3.1

Downregulated

J02874 Adipocyte lipid-binding protein �71.8 �4.1

Z49269 Chemokine HCC-1 �143.9 �2.9

L25876 Protein tyrosine phosphatase (CIP2) �69.5 �2.3

U51333 Hexokinase III (HK3) �108.0 �2.2

Genes of interest

Hs303649 MCP-1 �19.1 �1.3

M30257 VCAM-1 �2.0 �1.3

M37435 MCSF 3.0 �1.0

M93718 eNOS �73.3 �1.3

U91616 I�B� 3.8 1.2

D29675 Inducible NOS 13.7 1.1

U78678 Thioredoxin 46.0 �1.0

X91247 Thioredoxin reductase 351.7 1.5

M90100 Cyclooxygenase-2 10.4 0.7

A total of 6416 human genes were screened with GeneChip System(Affymetrix) before and after treatment with 5 �M HA for 16 hours.

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HO-1 Induction Preserves eNOS Expression AfterExposure to OxLDL and TNF-�To examine whether the antiatherogenic and vasculoprotec-tive effects of HO-1 are via the effect of eNOS expression, wepretreated HAECs with HA and observed the resultantexpression of eNOS after exposure to oxLDL and TNF-�(Figure 2). Untreated cells expressed a significant level ofeNOS protein, whereas there was little expression of HO-1mRNA and protein (Figure 2A, lane 1). Treatment withnative LDL (50 �g/mL) had little effect on eNOS expression(Figure 2A, lane 3), whereas treatment with oxLDL (50�g/mL) reduced eNOS expression accompanied by HO-1induction (Figure 2A, lane 4). Interestingly, HO-1 overex-pression by HA pretreatment significantly preserved eNOSexpression (Figure 2A, lane 5). The effect of HO-1 inductionon restoration of eNOS expression was abolished by cotreat-ment with SnPP IX (10 �mol/L), an HO inhibitor (Figure 2A,lane 6). Figure 2B shows the dose-dependent effect of HO-1on attenuation of eNOS expression by TNF-�. HO-1 induc-tion also recovered this attenuation.

To determine the HO-1 products responsible for preserva-tion of eNOS expression, cells were pretreated with either50 �mol/L [Ru(CO)3Cl2]2, a CO-releasing compound, or5 �mol/L bilirubin for 2 hours (Figure IIA, available online athttp://atvb.ahajournals.org), and then the cells were exposedto oxLDL (50 �g/mL) for 16 hours. Whereas [Ru(CO)3Cl2]2

treatment did not preserve eNOS expression, bilirubin treat-ment significantly preserved eNOS expression after exposureto oxLDL (Figure IIA). In addition, this effect was notinhibited by SB203580, an inhibitor of p38 mitogen-activatedprotein (MAP) kinase (Figure IIB).

HO-1 Induction Improves ImpairedEndothelium-Dependent Vascular RelaxationAfter we observed significant effects of HO-1 induction andbilirubin on the endothelial responses to oxLDL and TNF-�,we next examined whether these also affect endothelium-dependent vascular relaxation responses. To observe thechanges of endothelium-dependent vascular relaxation re-sponse, isolated thoracic aortic rings from LDL-receptorknockout mice were prepared (Figure 3A). Aortic rings frommice fed high-fat diet for 1 week (HF group) showedimpaired vasodilatory responses to acetylcholine comparedwith control (C group). Interestingly, aortic rings from micefed high-fat diet and pretreated with HA (HF�HA group)showed improved vasodilatory responses on high-fat HF diet.Bilirubin pretreatment (HF�BR group) resulted in similarimprovement, whereas CO exposure (HF�CO group) did nothave apparent effects (Figure 3A). In contrast, there were nosignificant differences among the 4 groups in endothelium-independent relaxation responses (Figure 3B). To examinewhether the improvement of vascular relaxation responses aremediated by restoration of eNOS by HA and bilirubintreatments, levels of HO-1 and eNOS expression in the aortawere examined (Figure 3C). After high-fat diet feeding,eNOS expression was significantly attenuated. In contrast,modest induction of HO-1 was observed. Importantly, HApretreatment augmented this HO-1 induction and restoredeNOS expression. Similar effects were confirmed after treat-

Figure 2. HO-1 induction by HA pretreatment reverses attenua-tion of eNOS expression by oxLDL (A) and TNF-� (B). Cellswere pretreated with 10 �mol/L HA for 16 hours or SnPP IX for2 hours. A, Cells were exposed to native LDL (50 �g/mL) oroxLDL (50 �g/mL). HO-1 mRNA expression was examined byNorthern blotting 6 hours after exposure to native LDL oroxLDL. HO-1 and eNOS proteins were examined by Westernblotting 16 hours after exposure to native LDL or oxLDL. B,HO-1 induction by HA pretreatment reversed attenuation ofeNOS by TNF-�. Cells were pretreated with 1, 5, and 10 �mol/LHA for 16 hours. Then cells were exposed to TNF-� (2 ng/mL)for another 16 hours. Changes of HO-1 and eNOS proteinexpression were examined by Western blotting. Densitometricanalysis of eNOS protein is shown. Data shown are representa-tive for 3 independent experiments. *P�0.001; **P�0.005.

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ment with bilirubin, whereas CO exposure did not haveapparent effects.

Next, we examined the levels of plasma lipid hydroperox-ides in these mice because we have observed that HO-1 hasinhibitory effects on lipid peroxidation in atheroscleroticanimal models.11,12 Significant reduction of plasma lipidhydroperoxides was observed in mice treated with HA andbilirubin (Figure 3D). There were no significant differencesin total plasma cholesterol, triglyceride, and HDL concentra-tions among the groups fed high-fat diet (data not shown).

DiscussionIn this study, we examined whether vascular endothelialHO-1 expression is involved in the endothelial activation andinsufficiency elicited by proinflammatory stresses, which areconsidered to be events essential for atherogenesis. Ourresults suggest that there are conditions under which HO-1expression significantly suppresses production of proinflam-matory mediators and reverses impaired eNOS expression inresponse to oxLDL and TNF-�.

To induce HO-1 in cultured endothelial cells, we used lowconcentrations of HA instead of hemin chloride because arecent study showed that HA is less toxic.18 Indeed, treatmentwith micromolar concentrations of HA did not have anyinjurious effects. We first examined the changes in geneexpression in endothelial cells before and after treatment with5 �mol/L HA for 16 hours, the conditions used to induceHO-1. A cDNA microarray analysis revealed that HO-1 was

the gene most strongly upregulated, and that several othergenes that have an ARE element also responded positively tothis treatment (Table). Importantly, the microarray also re-vealed that HA treatment did not generate significant changesin other genes that may affect atherogenic processes invascular endothelium. For instance, transcriptional changesof adhesion molecules, growth factors, and redox-sensitivetranscription factors such as nuclear factor-�B were notsignificant (Table). Similarly, the eNOS gene considered tohave antiatherosclerotic properties was not significantly al-tered by HA treatment.

Recent studies provide evidence that HO-1 functions as anintrinsic defense system in cardiovascular disorders.10–12,19,20

The protective effects of HO-1 may be attributed to itsbiological actions in vascular endothelium because endothe-lial HO-1 induction by HA significantly attenuated produc-tion of VCAM-1, MCP-1, and MCSF (Figures 1 and I) andpreserved eNOS expression (Figures 2 and II) in response toTNF-� and oxLDL. The functional improvement of theendothelium-dependent vasodilatory response was also con-firmed (Figure 3).

Cytoprotective effects of HO-1 have been observed re-cently in many kinds of cells, such as cardiomyocytes,20

hepatocytes,21 and fibroblasts,22 as well as in vascular endo-thelial cells.8 In this study, we tried to examine the biologicaleffects of the products of HO-1 in vascular endothelium. Toobserve the direct effects of CO, we treated cells with50 �mol/L ([Ru(CO)3Cl2]2), a CO-releasing compound, anddid not observe apparent biological effects, suggesting thatCO is not an HO-1 product that protects endothelium (FigureIIA), although recent studies have suggested there are multi-potent actions of CO in endothelium.23,24 However, it ispossible that CO produced from endothelium may regulateother cells in the artery wall. CO from endothelial cells mayfunction to relax smooth muscle cells under conditions ofinsufficient NO bioavailability and suppress reactive oxygenspecies generation in macrophages infiltrated into the arterywall. Indeed, this is supported by a recent report that COsuppresses proinflammatory cytokine production via inhibi-tion of the c-Jun N-terminal kinase pathway inmacrophages.25

In this study, protective effects on vascular endotheliumwere observed with another HO-1 product, bilirubin. Aphysiological concentration of bilirubin suppressed activationof proinflammatory genes such as VCAM-1, MCP-1, andMCSF (Figures 1 and I), restored eNOS expression (Figures2 and II), and improved endothelium-dependent vascularrelaxation responses (Figure 3). Bilirubin seems to alleviatethe effects of oxidative stresses on endothelium, which isconsidered to be a central factor in atherosclerosis. Becausethe anti-inflammatory action of CO in macrophages has beenshown recently to be mediated via the p38MAP kinasepathway,26 we examined the effect of SB203580, an inhibitorof p38MAP kinase, on the action of bilirubin (Figure IIB).However, inhibition of the p38MAP kinase pathway did notabolish the effect of bilirubin. Thus, bilirubin seems to relieveendothelial dysfunction independent of this pathway. Previ-ous studies have also indicated that bilirubin is a potentinhibitor of monocyte adhesion27 and chemotaxis9 to vascular

Figure 3. HO-1 induction by HA and bilirubin pretreatmentsimprove endothelium-dependent vascular relaxation response (Aand B), restore eNOS expression (C), and reduce plasma lipidhydroperoxides (D) in the LDL receptor knockout mice fed high-fat diet. Endothelium-dependent vascular relaxation responsesto acetylcholine (Ach; 10�9�10�5 mol/L; A) and endothelium-independent vascular relaxation responses sodium nitroprusside(SNP; 10�9�10�5 mol/L; B) were examined with aortic rings.Aortic rings from mice fed chow diet (CH; F), fed high-fat diet(HF; f), fed high-fat diet and pretreated with HA (HF�HA; E),fed high-fat diet and pretreated with CO (HF�CO; �), and fedhigh-fat diet and pretreated with bilirubin (HF�BR; �). Samplesfrom 6 mice in each group were analyzed. *P�0.01. C, Changesof HO-1 and eNOS protein expression in the aorta of LDLreceptor knockout mice; Western blotting. D, Changes inplasma lipid hydroperoxides in the LDL receptor knockout mice.Duplicate samples from 6 mice in each group were analyzed.*P�0.01; **P�0.05.

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endothelium. Our study indicates that eNOS is one of themolecular targets of bilirubin in vascular endothelium, andthat bilirubin functionally improves vasodilatory responses,although protective effects of bilirubin to other proinflamma-tory stimuli such as lipopolysaccharides and angiotensin IIshould also be explored (Figure 3). In conclusion, this studyrevealed that the antiatherosclerotic properties of HO-1 aremediated through effects on vascular endothelium and thatthese effects are predominantly caused by bilirubin.

AcknowledgmentsThis work was supported by a grant-in-aid (14570682 and16590703) from the Ministry of Education, Science, and Cultureof Japan.

References1. Libby P. Inflammation in atherosclerosis. Nature. 2002;420:868–874.2. Khan BV, Parthasarathy SS, Alexander RW, Medford RM. Modified low

density lipoprotein and its constituents augment cytokine-activatedvascular cell adhesion molecule-1 gene expression in human vascularendothelial cells. J Clin Invest. 1995;95:1262–1270.

3. Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ.Absence of monocyte chemoattractant protein-1 reduces atherosclerosisin low density lipoprotein receptor-deficient mice. Mol Cell. 1998;2:275–281.

4. Smith JD, Trogan E, Ginsberg M, Grigaux C, Tian J, Miyata M.Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E. Proc Natl Acad Sci U S A.1995;92:8264–8268.

5. Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD. Impairmentof endothelium-dependent arterial relaxation by lysolecithin in modifiedlow-density lipoproteins. Nature. 1990;344:160–162.

6. Maines MD. The heme oxygenase system: a regulator of second mes-senger gases. Annu Rev Pharmacol Toxicol. 1997;37:517–554.

7. Poss KD, Tonegawa S. Reduced stress defense in heme oxygenase1-deficient cells. Proc Natl Acad Sci U S A. 1997;94:10925–10930.

8. Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K,Kasahara Y, Koizumi S. Oxidative stress causes enhanced endothelial cellinjury in human heme oxygenase-1 deficiency. J Clin Invest. 1999;103:129–135.

9. Ishikawa K, Navab M, Leitinger N, Fogelman AM, Lusis AJ. Inductionof heme oxygenase-1 inhibits the monocyte transmigration induced bymildly oxidized LDL. J Clin Invest. 1997;100:1209–1216.

10. Wang LJ, Lee TS, Lee FY, Pai RC, Chau LY. Expression of hemeoxygenase-1 in atherosclerotic lesions. Am J Pathol. 1998;152:711–720.

11. Ishikawa K, Sugawara D, Wang X, Suzuki K, Itabe H, Maruyama Y,Lusis AJ. Heme oxygenase-1 inhibits atherosclerotic lesion formation inLDL-receptor knockout mice. Circ Res. 2001;88:506–512.

12. Ishikawa K, Sugawara D, Goto J, Watanabe Y, Kawamura K, Shiomi M,Itabe H, Maruyama Y. Heme oxygenase-1 inhibits atherogenesis inWatanabe heritable hyperlipidemic rabbits. Circulation. 2001;104:1831–1836.

13. Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, GreenCJ. Carbon monoxide-releasing molecules: characterization of bio-chemical and vascular activities. Circ Res. 2002;90:E17–E24.

14. Takabe W, Mataki C, Wada Y, Ishii M, Izumi A, Aburatani H, HamakuboT, Niki E, Kodama T, Noguchi N. Gene expression induced by BO-653,probucol and BHQ in human endothelial cells. J Atheroscler Thromb.2000;7:223–230.

15. Ishii M, Hashimoto S, Tsutsumi S, Wada Y, Matsushima K, Kodama T,Aburatani H. Direct comparison of GeneChip and SAGE on the quanti-tative accuracy in transcript profiling analysis. Genomics. 2000;68:136–143.

16. Yagi K. Simple procedure for specific assay of lipid hydroperoxides inserum or plasma. Methods Mol Biol. 1998;108:107–110.

17. Chen XL, Varner SE, Rao AS, Grey JY, Thomas S, Cook CK, Was-serman MA, Medford RM, Jaiswal AK, Kunsch C. Laminar flowinduction of antioxidant response element-mediated genes in endothelialcells. A novel anti-inflammatory mechanism. J Biol Chem. 2003;278:703–711.

18. Balla J, Balla G, Jeney V, Kakuk G, Jacob HS, Vercellotti GM. Ferri-porphyrins and endothelium: a 2-edged sword-promotion of oxidationand induction of cytoprotectants. Blood. 2000;95:3442–3450.

19. Yet SF, Layne MD, Liu X, Chen YH, Ith B, Sibinga NE, Perrella MA.Absence of heme oxygenase-1 exacerbates atherosclerotic lesion for-mation and vascular remodeling. FASEB J. 2003;17:1759–1761.

20. Araujo JA, Meng L, Tward AD, Hancock WW, Zhai Y, Lee A, IshikawaK, Iyer S, Buelow R, Busuttil RW, Shih DM, Lusis AJ, Kupiec-WeglinskiJW. Systemic rather than local heme oxygenase-1 overexpressionimproves cardiac allograft outcomes in a new transgenic mouse.J Immunol. 2003;171:1572–1580.

21. Amersi F, Shen XD, Anselmo D, Melinek J, Iyer S, Southard DJ, KatoriM, Volk HD, Busuttil RW, Buelow R, Kupiec-Weglinski JW. Ex vivoexposure to carbon monoxide prevents hepatic ischemia/reperfusioninjury through p38 MAP kinase pathway. Hepatology. 2002;35:815–823.

22. Dennery PA, Sridhar KJ, Lee CS, Wong HE, Shokoohi V, Rodgers PA,Spitz DR. Heme oxygenase-mediated resistance to oxygen toxicity inhamster fibroblasts. J Biol Chem. 1997;272:14937–14942.

23. Thom SR, Fisher D, Xu YA, Notarfrancesco K, Ischiropoulos H.Adaptive responses and apoptosis in endothelial cells exposed to carbonmonoxide. Proc Natl Acad Sci U S A. 2000;97:1305–1310.

24. Abraham NG, Kushida T, McClung J, Weiss M, Quan S, Lafaro R,Darzynkiewicz Z, Wolin M. Heme oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel endo-thelial cells. Circ Res. 2003;93:507–514.

25. Morse D, Pischke SE, Zhou Z, Davis RJ, Flavell RA, Loop T, OtterbeinSL, Otterbein LE, Choi AM. Suppression of inflammatory cytokineproduction by carbon monoxide involves the JNK pathway and AP-1.J Biol Chem. 2003;278:36993–36998.

26. Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, Wysk M, Davis RJ,Flavell RA, Choi AM. Carbon monoxide has anti-inflammatory effectsinvolving the mitogen-activated protein kinase pathway. Nat Med. 2000;6:422–428.

27. Hayashi S, Takamiya R, Yamaguchi T, Matsumoto K, Tojo SJ, TamataniT, Kitajima M, Makino N, Ishimura Y, Suematsu M. Induction of hemeoxygenase-1 suppresses venular leukocyte adhesion elicited by oxidativestress: role of bilirubin generated by the enzyme. Circ Res. 1999;85:663–671.

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*

MCP-1 (pg/ml)

B

0

100

200

300*

0TNF-α - + ++++

HA (µM) 0 10 10 0 0

SnPP IX - - --+-

[Ru(CO)3Cl2]2Bilirubin - - +---- - -+--

* **A **

0

100

200

300

400

MCP-1 (pg/ml)

0oxLDL - + ++++

HA (µM) 0 10 10 0 0

SnPP IX - - --+-

[Ru(CO)3Cl2]2Bilirubin - - +---- - -+--

0oxLDL - + ++++

HA (µM) 0 10 10 0 0

SnPP IX - - --+-

[Ru(CO)3Cl2]2 - - +---- - -+--

0oxLDL - + ++++

HA (µM) 0 10 10 0 0

SnPP IX - - --+-

- - +---- - -+--

* *

C **

0

100

200

300 * *

MCSF (pg/ml)

0oxLDL - + ++++

HA (µM) 0 10 10 0 0

SnPP IX - - --+-

[Ru(CO)3Cl2]2Bilirubin - - +---- - -+--

*

100

200

300D **

MCSF (pg/ml)

*

0

TNF-α - + ++++HA (µM) 0 10 10 0 0

SnPP IX - - --+-

[Ru(CO)3Cl2]2Bilirubin - - +---- - -+--

0

****

eNOS

+[Ru(CO)3Cl2 ] 2

Bilirubin

+oxLDL ---

+ +--

+ --

A

B

eNOS

oxLDL

Bilirubin

SB203580

---

+--

++- +

++ -

+-

-

+-

Online-Figure Legends

Fig. I. HO-1 induction by HA and bilirubin pretreatment attenuates MCP-1 and MCSF

production caused by oxLDL and TNFα in HAEC. Cells were pretreated with 1, 5 and 10 µM

HA for 16 hours or with SnPP IX (10 µM), [Ru(CO)3Cl2]2 (50 µM), and bilirubin (5 µM) for 2

hours. Then, cells were exposed to oxLDL (50 µg/ml) (A, C) or TNFα (2 ng/ml) (B, D) for 3

hours, washed, incubated in fresh media for another 3 hours. MCP-1 (A, B) and MCSF (C, D)

production (pg/ml) were analyzed by ELISA with conditioned media in duplicate. Data shown

are representative for three (A, C) and four (B, D) independent experiments. *; p<0.001, **;

p<0.005.

Fig. II. Preservation of eNOS expression after exposure to oxLDL is not attributed to CO but to

bilirubin. (A, B) Cells were pretreated with either 50 µM [Ru(CO)3Cl2]2 or with 5 µM bilirubin for

2 hours. Then, cells were exposed to oxLDL (50 µg/ml) for 16 hours. Cells pretreated with

[Ru(CO)3Cl2] 2 were exposed to a cold light to release CO. eNOS protein expression were

examined by Western blotting. SB203580, an inhibitor of p38 MAP kinase, was added 2 hours

before bilirubin pretreatment. Data shown are representative for three independent experiments.