. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Increased serum high-mobility group box-1 andcleaved receptor for advanced glycationendproducts levels and decreased endogenoussecretory receptor for advanced glycationendproducts levels in diabetic and non-diabeticpatients with heart failureLing Jie Wang1,2†, Lin Lu1,2†, Feng Ru Zhang1†, Qiu Jing Chen2, Raffaele De Caterina3*,and Wei Feng Shen1,2*

1Department of Cardiology, Rui Jin Hospital, Shanghai Jiaotong University School of Medicine, 197 Rui Jin Road II, Shanghai 200025, People’s Republic of China; 2Institute ofCardiovascular Diseases, Shanghai Jiaotong University School of Medicine, Shanghai 200025, People’s Republic of China; and 3University Cardiology Department, “G. d’Annunzio”University—Chieti, Ospedale SS. Annunziata Via dei Vestini, I-66013 Chieti, Italy

Received 25 April 2010; revised 25 September 2010; accepted 30 September 2010; online publish-ahead-of-print 24 January 2011

Aims High-mobility group box-1 (HMGB1) is a ligand for the receptor for advanced glycation endproducts (RAGE). AnHMGB1–RAGE interaction has been implicated in cardiac dysfunction. We assessed the association of HMGB1and RAGE isoforms with heart failure (HF) in diabetic and non-diabetic patients.

Methodsand results

We assayed serum levels of HMGB1, cleaved RAGE (cRAGE), endogenous secretory RAGE (esRAGE), high-sensi-tivity C-reactive protein (hsCRP), and N-terminal pro-brain natriuretic peptide (NT-proBNP) in parallel with assess-ment of left ventricular volumes and function in 125 diabetic and 222 non-diabetic Chinese patients with chronic HF.Of the total, 79 diabetic patients without HF and 220 normal subjects served as diabetic and normal controls,respectively. Serum HMGB1, cRAGE, hsCRP, and NT-proBNP levels were higher and, in contrast, esRAGE levelslower in HF patients than in subjects without HF (for all; P , 0.01), with higher levels of cRAGE and hsCRP in diabeticHF vs. non-diabetic HF patients (P , 0.01). For HF patients—with or without diabetes—HMGB1 levels correlatedpositively with left ventricular end-diastolic and end-systolic volumes (r ¼ 0.267 and r ¼ 0.321, respectively) andNT-proBNP values (r ¼ 0.497), and were inversely related to ejection fraction (r ¼ 20.461; all P , 0.001). SerumcRAGE levels correlated with NT-proBNP values (r ¼ 0.451) and New York Heart Association functional class(r ¼ 0.402; both P , 0.001). Multivariable regression analysis revealed that HMGB1, cRAGE, and esRAGE were con-sistently associated with HF in diabetic and non-diabetic patients.

Conclusion Heart failure patients have increased serum HMGB1 and cRAGE and decreased esRAGE levels, and these are relatedto the severity of HF in both diabetic and non-diabetic patients. Such associations are worth further investigation.

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Keywords HMGB1 † esRAGE † cRAGE † Chronic heart failure † Diabetes

† These authors contributed equally to this manuscript.

* Corresponding author. Fax: +86 021 64457177, Email: rjshenweifeng@yahoo.com.cn (W.F.S.); Tel: +39 0871 41512, Fax: +39 0871 402817, Email: rdecater@unich.it (R.D.C.)

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2011. For permissions please email: journals.permissions@oup.com.

European Journal of Heart Failure (2011) 13, 440–449doi:10.1093/eurjhf/hfq231

IntroductionInteraction of advanced glycation endproducts (AGEs) with themain receptor (RAGE), and subsequent intracellular signalling,has been implicated in diabetic complications.1 Advanced glycationendproducts, acting as pro-inflammatory triggers, are associatedwith endothelial dysfunction and coronary artery disease, in bothdiabetic 2– 4 and non-diabetic patients.5 Interestingly, AGEs havealso been recently implicated in cardiac dysfunction,6– 9 thus emer-ging as a risk factor for poor clinical outcomes in chronic heartfailure (HF).10,11

Besides full-length membrane-bound RAGE, two majoradditional isoforms have been recently identified, namely theendogenous secretory RAGE (esRAGE) and a cleaved form ofRAGE (cRAGE).12 Endogenous secretory RAGE, a quantitativelyminor isoform, is generated through alternative splicing ofpre-mRNA; cRAGE is proteolytically cleaved from the cellsurface by matrix metalloproteinases, and then shed into thebloodstream. Both variants may act as decoy ligands for AGEsand several inflammatory cytokines. Recent studies have,however, suggested a reciprocal relationship between these twoligands, whereby decreased esRAGE and/or increased cRAGElevels are biomarkers of heightened ligand–RAGE interaction indiabetes, atherosclerosis, and other inflammatory diseases, poss-ibly underscoring an inadequate endogenous protectiveresponse.13 This suggestion has been recently extended to HF,where esRAGE levels are considered a negative prognosticfactor.14

High-mobility group box-1 (HMGB1) is a nuclear DNA-bindingprotein, passively released from necrotic cells and actively secretedby activated immune cells.15 Recently, HMGB1 has been demon-strated to be a novel ligand for RAGE, cRAGE, and esRAGE.16,17

The engagement of HMGB1 with RAGE promotes the sheddingof the receptor.18 Its signalling via RAGE activates inflammatorypathways and intensifies cellular oxidative stress, which results inprofuse production of inflammatory cytokines and elevatedexpression of adhesion molecules.17 Increased HMGB1 levels areassociated with ischaemia–reperfusion injury in mice, and withcoronary artery disease in diabetic patients,16,19 and are alsoinvolved in post-infarction inflammatory response and left ventri-cular remodelling.20 On the other hand, low levels of HMGB1have also distinctly favourable biological properties, being capableof attracting stem cells,21 facilitating myocardial cell regenerationand differentiation,22 enhancing angiogenesis, and consequentlyimproving myocardial function and survival after myocardial infarc-tion.23 Indeed, low-grade inflammation elicited by intra-myocardialinjection of HMGB1 can favour the recovery of chronic post-infarction cardiac remodelling and limit HF.24

Research over the last two decades has provided some cluesabout the association of myocardial dysfunction with inflammation,increased cytokine production, and fibrous tissue deposition.Inflammatory activation and increased cytokine production, thus,apparently play some role in the clinical development and pro-gression of HF.25 Based on these findings, we hypothesized thatHMGB1 concentrations may mark—and perhaps contribute to—the development of HF, with accompanying esRAGE and cRAGEchanges. We predicted a graded relationship between HF severity

and changes in plasma levels of HMGB1 and related proteins. Wetherefore evaluated the association of serum concentrations ofHMGB1 (primary objective), as well as esRAGE and cRAGE,together with levels of high-sensitivity C-reactive protein(hsCRP) as a marker of inflammation and N-terminal precursorof brain natriuretic peptide (NT-proBNP) (secondary objectives)as a HF biomarker, in non-diabetic and type 2 diabetic patientswith chronic HF.

MethodsThe study protocol was approved by the local hospital Ethics Commit-tee, and written informed consent was obtained from all participants.

Study populationWe restricted the inclusion to Chinese patients of Han nationality withsystolic chronic HF due to ischaemic or idiopathic dilated cardiomyo-pathy. The study, therefore, included 149 consecutive patients withischaemic HF (mean age: 68.3+ 9.9 years; duration of disease: 1.5+2.1 years), and 198 patients with HF caused by idiopathic dilated car-diomyopathy (mean age: 57.0+13.5 years; duration of disease: 2.9+3.3 years) recruited between January 2000 and May 2006 from threeteaching hospitals affiliated to the Shanghai Jiaotong UniversitySchool of Medicine in China. Systolic HF was diagnosed according tothe European Society of Cardiology guidelines, including patientswith symptoms or signs of HF and left ventricular ejection fraction(LVEF) ,45%, assessed by echocardiography. For patients withidiopathic dilated cardiomyopathy and cardiovascular risk factors(e.g. with associated type 2 diabetes), the presence of significant cor-onary artery disease was excluded by coronary angiography. Of the347 patients with HF, 222 were non-diabetic and 125 had type 2 dia-betes. Diabetes was defined according to the American DiabetesAssociation criteria as: two fasting plasma glucose levels ≥7.0 mmol/L, or symptoms of diabetes plus a casual post-prandial plasmaglucose reading ≥11.1 mmol/L, or a 2 h glucose reading≥11.1 mmol/L after a 75 g glucose load, or taking oral hypoglycaemicdrugs or parenteral insulin. To avoid confounding variables, weexcluded patients with a history of viral myocarditis, hypertrophic car-diomyopathy, primary valvular disease, or pulmonary heart disease.We also excluded patients with type 1 diabetes, chronic viral or bac-terial infections, tumours, or immune disorders. Detailed informationwas obtained on demographics, clinical manifestation, and medications,as well as New York Heart Association (NYHA) functional class, andechocardiographic measurements were used to evaluate HF severity.

Two hundred and twenty normal Chinese subjects (132 men and 88women; mean age: 60+13 years) and 79 Chinese patients with type 2diabetes only (37 men and 42 women; mean age: 64+ 10 years)served as normal and diabetic controls, respectively. Their gender dis-tribution was the same as in the general population; age was purposelymatched between controls and the HF population. Detailed medicaland family history was taken, and fasting blood samples were collectedduring an annual physical check-up. In the normal controls, serumlevels of glucose, lipid profiles, liver and renal function tests, and theelectrocardiogram were normal in all subjects, and none had ahistory of cardiovascular diseases (including past history of angina/myo-cardial infarction). In the diabetic control group, we excluded patientswith prior coronary heart disease, stroke, or severe renal dysfunction.

Biochemical investigationsPeripheral venous blood samples were collected after an overnightfast. Serum glucose, blood urea nitrogen, creatinine, uric acid, total

HMGB1 and RAGE in heart failure 441

cholesterol, low-density lipoprotein cholesterol, high-density lipopro-tein cholesterol, lipoprotein (a), apolipoprotein A, apolipoprotein B,and triglycerides were measured using standard laboratory techniqueson a Hitachi 912 Analyser (Roche Diagnostics, Mannheim, Germany).Serum NT-proBNP was determined using a commercially availableelectrochemiluminescence immunoassay kit (Roche Diagnostics).Serum HMGB1 levels were assessed with an enzyme-linked immuno-sorbent assay (ELISA) kit (HMGB1 ELISA kit II; Shino-Test Corpor-ation, Tokyo, Japan) according to the manufacturer’s instructions.The detection limit for HMGB1 was 0.2 ng/mL, with an inter-assaycoefficient of variation ,10%. Levels of cRAGE and esRAGE werealso measured using ELISA kits (Quantikine; R&D Systems, MN,USA; B-Bridge International, Mountain View, CA, USA, respectively).

Echocardiographic assessmentTransthoracic two-dimensional echocardiography was performedusing a Hewlett-Packard Sonos 2500 (Hewlett-Packard, San Diego,CA, USA) or a GE Vivid-7 system (General Electric Vingmed SoundAS, Horton, Norway) equipped with 2.5 or 1.7/3.4 MHz transducers,respectively. Images were obtained at rest with the patient lying inthe left lateral decubitus position at end-expiration. Left ventricularend-diastolic and end-systolic volumes were measured according tothe biplane Simpson’s method based on the American Society of Echo-cardiography recommendations, and LVEF was calculated. An averageof three consecutive cardiac cycles was used for each patient.

Follow-upAll HF patients were prescribed standard HF treatments and wereseen every 1–3 months in a dedicated HF clinic. During each visit,heart rate, blood pressure, and new clinical manifestations wererecorded; echocardiography was performed every 6 months.Adverse events (hospitalization or death from HF) were recordedduring each visit or by telephone with patients or family members.Hospitalization for HF was defined as that due to progressive fluidretention and the need to increase or change medications. Twotrained physicians independently reviewed all medical notes, includingforms from visits to the emergency department and hospital medicalrecords.

Statistical analysisContinuous variables are presented as mean+ standard deviation(SD). Differences between groups were compared with two-factor(HF, diabetes) analysis of variance followed by Dunnett post hocbetween-group analysis or by the non-parametric Kruskal–Wallistest. Categorical data were summarized as frequencies or percentages,and differences between groups were evaluated by the x2 test. Thesample size (347 patients with HF and 299 subjects without HF, withor without diabetes) was such to yield .80% power to detect differ-ences between HF and non-HF patients for HMGB1, NT-proBNP,cRAGE, and hsCRP (for all, 99% power both in non-diabetic and dia-betic patients), as well as esRAGE levels (99% in non-diabetic and 87%in diabetic patients, respectively) under a type I error probability of0.05 (12a) for a two-sided test.

Correlations of serum HMGB1 (on a logarithmic scale) with otherbiomarkers or echocardiographic measurements (left atrial diametersand ventricular volumes) were assessed by Pearson’s test, and associ-ations of these biomarkers with LVEF (not normally distributed) orNYHA functional class (categorical variable) by Spearman’s test. Thelinear correlation of LVEF with HMGB1 was still represented withthe Pearson’s method to represent their crude distributions. Weused two models in multivariable logistic regression analysis for the

presence of HF in non-diabetic and diabetic subjects, respectively. InModel I, multivariable adjustment was made for conventional riskfactors measured at baseline examination, and included age, gender,smoking, hypertension, systolic/diastolic blood pressure, triglycerides,total cholesterol, blood urea nitrogen, creatinine, uric acid, fastingglucose, and glycated haemoglobin (by a conditional logistic regressionmethod). In Model II, the multivariable-adjusted odd ratios (ORs) andtheir 95% confidence intervals (CI) for HF associated with the bio-markers of interest were compared with the respective normal- anddiabetic-matched controls, synchronously estimated together with sig-nificantly independent conventional risk factors established in Model I(by the backward conditional logistic regression method). In addition,the ORs were given for a 1- or 1/2-SD increase of each biomarker,blood pressure, creatinine, and uric acid in control group.

All statistical analyses were done using the SPSS version 13.0 soft-ware (SPSS, Inc., Chicago, IL, USA).

Results

Clinical characteristicsThe diabetic and non-diabetic patients with HF were more fre-quently male and cigarette smokers than the normal or diabeticcontrols. Compared with non-diabetic patients with HF, thosewith both HF and diabetes were older and had a higher ratio ofischaemic vs. non-ischaemic aetiology, a poorer renal function,and more abnormal lipid profiles. No significant differencesbetween HF patients with and without diabetes were observedfor left ventricular end-diastolic or end-systolic volumes or LVEF,with the exception of a larger left atrial size in diabetic patientswith HF. During a 2-year follow-up, 22 out of 125 (17.6%) diabeticand 23 out of 222 (10.4%) non-diabetic patients with HF died ofsudden cardiac death or refractory HF (Table 1).

Influence of heart failure and its aetiologyon biological analytesSerum levels of HMGB1(7.57+8.70 vs. 2.66+4.22 ng/mL), cRAGE(1005.7+1176.3 vs. 594.5+401.5 pg/mL), hsCRP (16.80+17.08vs. 7.83+6.67 mg/L), and NT-proBNP (3509.9+5206.8 vs.201.9+567.5 pg/mL) were higher, but esRAGE levels (241.5+243.0 vs. 345.5+146.3 pg/mL) were lower in the overall populationof patients with HF than in thosewithout HF (all P , 0.01), while elev-ated levels of cRAGE (643.5+1176.3 vs. 594.5+401.5 pg/mL indiabetic and non-diabetic subjects, respectively), hsCRP (11.39+9.31 vs. 5.15+4.38 mg/L; both P , 0.05), and reduced esRAGElevels (253.5+109.9 vs. 415.3+131.3 pg/mL; P , 0.001) werepresent in association with diabetes in patients without HF. In otherwords, levels of HMGB1 and NT-proBNP, in contrast to the otheranalytes assayed, were selectively influenced by the presence of HF.Higher levels of cRAGE and hsCRP, but not of HMGB1 oresRAGE, were observed in diabetic vs. non-diabetic patients withHF (for all; P , 0.01; Figure 1 and Table 2). In addition, esRAGElevels were selectively related to the aetiology of HF, with higherlevels in dilated vs. ischaemic cardiomyopathy (260.5+290.3 vs.222.3+182.0 pg/mL; P ¼ 0.015); whereas cRAGE levels were influ-enced by both the aetiology of HF (P ¼ 0.015) and diabetes (P ¼0.003), with the highest levels seen in diabetic patients with dilatedcardiomyopathy (1743.8+1838.0 pg/mL), and the lowest levels in

L.J. Wang et al.442

non-diabetic patients with ischaemic cardiomyopathy (752.5+804.6 pg/mL). However, HMGB1 levels were not influenced byaetiology (5.38+0.69 vs. 6.91+0.72 ng/mL in ischaemic anddilated cardiomyopathy, respectively; P ¼ 0.066) or the presence ofdiabetes (6.14+0.79 vs. 6.10+0.60 ng/mL in diabetic and non-diabetic patients, respectively; P ¼ 0.123) in HF (Table 3).

Serum HMGB1 levels correlated positively with hsCRP(Pearson’s r ¼ 0.208; P , 0.001) and cRAGE values (Pearson’sr ¼ 0.086; P ¼ 0.042), and were inversely related toesRAGE (Pearson’s r ¼ 20.203; P , 0.001). Serum esRAGEcorrelated negatively with hsCRP levels (Pearson’s r ¼ 20.249;P , 0.001).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 1 Baseline clinical characteristics and biochemical assessments of patients studied

Non-diabetic patients Diabetic patients

No HF (n 5 220) HF (n 5 222) P-value No HF (n 5 79) HF (n 5 125) P-value

Male/female (n) 132/88 167/55 37/42 94/31

Age (years) 59.5 (13.2) 60.4 (14.5) 0.55 64.1 (10.4)** 64.4 (10.4)‡,§ 0.137

Aetiology (ischaemic/dilated cardiomyopathy, n) 68/154 81/44§

Hypertension [n (%)] 25 (11.4) 94 (42.3) ,0.001 53 (67.4) 94 (75.4)} 0.208

Cigarette smoking [n (%)] 19 (14.4) 101 (45.4) ,0.001 16 (20.3) 48 (38.6) 0.007

NYHA functional class II/III/IV (n) 109/107/6 29/85/11}

Systolic blood pressure (mmHg) 123.1 (18.5) 124.5 (20.4) 0.53 136.0 (19.5) 129.1 (20.4)‡,} 0.080

Diastolic blood pressure (mmHg) 75.2 (10.2) 77.1 (11.9) 0.215 80.1 (10.6) 79.0 (11.2)† 0.342

Heart rate (b.p.m.) 71.6 (4.7) 77.8 (16.1) 0.184 77.1 (9.7) 77.8 (13.1) 0.954

NT-proBNP (pg/mL) 157.9 (195.3) 3297.4 (5409.8) ,0.001 172.1 (361.8) 2659.0 (3302.7)‡ ,0.001

Left atrial diameter (mm) 37.5 (4.8) 45.0 (6.3) 0.007 40.0 (6.8) 46.9 (7.1)‡,§ ,0.001

Left ventricular end-diastolic volume (mL) 107.3 (27.0) 294.3 (122.9) ,0.001 104.3 (16.8) 276.0 (134.6)‡ ,0.001

Left ventricular end-systolic volume (mL) 37.4 (12.7) 158.1 (84.8) ,0.001 35.5 (10.8) 151.4 (99.1)‡ ,0.001

Ejection fraction (%) 63.3 (5.9) 36.0 (6.9) ,0.001 65.1 (6.1) 36.6 (7.6)‡ ,0.001

Fasting glucose (mmol/L) 4.73 (0.50) 5.27 (1.16) 0.001 6.91 (2.38)** 6.66 (2.32)‡,§ 0.036

Glycated haemoglobin (HBA1c, %) 5.85 (0.35) 5.99 (0.52) 0.863 7.88 (1.27)** 7.30 (1.24)‡,§ 0.001

Blood urea nitrogen (mmol/L) 5.04 (1.22) 6.93 (3.18) ,0.001 5.81 (1.78) 7.60 (2.72)‡ 0.003

Creatinine (mmol/L) 79.1 (17.3) 104.6 (70.5) ,0.001 84.2 (21.9) 104.8 (42.5)‡ ,0.001

Uric acid (mmol/L) 310.5 (65.0) 395.3 (120.5) ,0.001 324.9 (72.9) 394.8 (134.2)‡ ,0.001

Triglycerides (mmol/L) 1.09 (0.50) 1.53 (0.96) ,0.001 2.09 (1.33)** 1.64 (1.02)‡ ,0.001

Total cholesterol (mmol/L) 4.62 (0.65) 4.31 (0.98) ,0.001 4.82 (1.12) 4.21 (0.98)‡ ,0.001

High-density lipoprotein cholesterol (mmol/L) 1.42 (0.28) 1.21 (0.45) ,0.001 1.20 (0.29) 1.07 (0.30)‡,} 0.020

Low-density lipoprotein cholesterol (mmol/L) 2.79 (0.58) 2.50 (0.78) 0.011 2.80 (0.86) 2.52 (0.81)† 0.025

Apoprotein A (g/L) 1.24 (0.17) 1.15 (0.21) ,0.001 1.26 (0.19) 1.09 (0.23)‡ 0.001

Apoprotein B (g/L) 0.78 (0.15) 0.85 (0.19) 0.041 0.95 (0.25) 0.85 (0.20)† 0.020

Lipoprotein (a) (g/L) 0.19 (0.14) 0.24 (0.21) 0.112 0.19 (0.16) 0.19 (0.18) 0.314

Angiotensin-converting enzyme-inhibitors [n (%)] 162 (72.8) 73 (58.8) 0.022

Angiotensin receptor blockers [n (%)] 52 (23.4) 43 (34.7) 0.035

b-Blockers [n (%)] 209 (94.2) 113 (90.1) 0.193

Nitrates [n (%)] 107 (48.1) 93 (74.3) ,0.001

Statins [n (%)] 61 (27.7) 67 (53.5) ,0.001

Diuretics [n (%)] 167 (75.2) 88 (70.7) 0.400

Aspirin [n (%)] 183 (80.7) 117 (90.8) 0.018

Digoxin [n (%)] 160 (72.3) 83 (66.3) 0.280

Hospitalization during the 2-year follow-up [n (%)] 99 (44.6) 67 (53.6) 0.107

Death during the 2-year follow-up [n (%)] 23 (10.4) 22 (17.6) 0.054

Values are given as mean (standard deviation) or number (percentage).HF, heart failure.**P , 0.01, diabetic patients with no HF vs. normal controls [non-diabetic subjects without HF (no HF)].†P , 0.05.‡P , 0.01, diabetic patients with HF vs. normal controls.}P , 0.05.§P , 0.01, diabetic patients with HF vs. non-diabetic patients with HF.

HMGB1 and RAGE in heart failure 443

Association of biological analytes withheart failure severityThe relationship between serum measurements of HMGB1 levelsand HF severity is depicted in Figure 2. High-mobility group box-1was positively related to NT-proBNP (Pearson’s r ¼ 0.497; P ,

0.001), and left ventricular end-diastolic and end-systolic volumes(Pearson’s r ¼ 0.267 and r ¼ 0.321, respectively; both P , 0.001),but was inversely related to LVEF (Spearman’s r ¼ 20.461; P ,

0.001) in both diabetic and non-diabetic HF patients. Moreover,HMGB1 levels showed a positive correlation with NYHA functionalclass in HF diabetic patients (Spearman’s r ¼ 0.184; P ¼ 0.039), butnot in HF non-diabetic patients (Spearman’s r ¼ 0.074; P ¼ 0.291).

Similarly, serum levels of cRAGE were positively related toNT-proBNP levels independent of diabetes (Spearman’s r ¼0.451; P , 0.001), but were positively associated with left atrialdiameters (Pearson’s r ¼ 0.360; P , 0.001), left ventricular end-diastolic and end-systolic volumes (Pearson’s r ¼ 0.358 and r ¼0.328, respectively; both P , 0.001), and inversely related toLVEF (Spearman’s r ¼ 20.356; P , 0.001) only in diabetic patientswith HF. In addition, in both non-diabetic and diabetic patients withHF, serum cRAGE levels correlated significantly with NYHA func-tional class (Spearman’s r ¼ 0.326 and r ¼ 0.567, respectively; bothP , 0.001). There was a stepwise elevation of cRAGE levels with

worsening NYHA functional classes [median and 25–75% percen-tiles: 260 (83–663) vs. 381 (155–1160) pg/mL in class II; 995(519–2297) vs. 1085 (428–2733) pg/mL in class III; 5838 (3194–6489) vs. 2220 (802–5175) pg/mL in class IV, in non-diabetic vs.diabetic patients, respectively].

However, serum esRAGE levels correlated inversely withNT-proBNP levels (Pearson’s r ¼ 20.303; P , 0.001) only in non-diabetic patients, and with left atrial diameters (Pearson’sr ¼ 20.259; P ¼ 0.003) and left ventricular end-systolic volumes(Pearson’s r ¼ 20.175; P ¼ 0.048) only in diabetic patients withHF. No significant correlation between esRAGE and LVEF wasfound in either group (both P . 0.05). In addition, esRAGElevels were negatively related to NYHA functional class in diabeticand non-diabetic patients with HF (Spearman’s r ¼ 20.292(P ¼ 0.001) and r ¼ 20.161 (P ¼ 0.011), respectively). Thedetailed association between these biological analytes and HFseverity is shown in Table 4.

Multivariable logistic regression analysisfor the presence of heart failureMultivariable stepwise logistic regression analysis run in all subjects,with or without HF, revealed that, adjusting for traditionalcardiovascular risk factors (Model I), smoking (OR: 2.235, 95%:

Figure 1 Comparison of serum levels of high-mobility group box-1 (HMGB1), cleaved receptor for advanced glycation endproducts(cRAGE), endogenous secretory receptor for advanced glycation endproducts (esRAGE), high-sensitivity C-reactive protein (hsCRP), andN-terminal pro-brain natriuretic peptide (NT-proBNP) among diabetic and non-diabetic patients with or without HF.

L.J. Wang et al.444

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 2 Serum levels of high-mobility group box-1 and other biological analytes in diabetic and non-diabetic patients with or without heart failure

Non-diabetic patients Diabetic patients Diabetes HF Diabetes 3 HF

No HF (n 5 220) HF (n 5 222) P-value No HF (n 5 79) HF (n 5 125) P-value P1-value P2-value P3-value

HMGB1 (ng/mL) 1.84+2.27 6.31+7.58 ,0.001 2.41+3.46 5.99+8.24‡ ,0.001 0.423 ,0.001 0.446

cRAGE (pg/mL) 550.5+397.1 962.91+1155.5 0.001 666.1+355.1* 1159.6+1309.7‡, §§ ,0.001 0.035 ,0.001 0.044

esRAGE (pg/mL) 467.0+128.5 245.5+281.7 ,0.001 285.3+109.0** 237.2+180.0‡ 0.001 ,0.001 ,0.001 ,0.001

hsCRP (mg/L) 3.32+2.23 20.68+32.04 ,0.001 7.08+4.03* 33.40+23.20‡, § ,0.001 0.025 ,0.001 0.033

NT-proBNP (pg/mL) 157.9+195.3 3297.4+5409.8 ,0.001 172.1+361.8 2659.0+3302.7‡ ,0.001 0.411 ,0.001 0.531

Values are given as mean+ standard deviation. P1, P2, and P3 values stand for the contributions of diabetes, HF, and HF aetiology-diabetes interaction to the differences of biological analytes among the four groups by two-way analysis ofvariance, respectively.*P , 0.05.**P , 0.01, diabetic patients with no HF vs. normal controls (non-diabetic subjects with no HF).‡P , 0.01, diabetic patients with HF vs. normal controls.§P , 0.05.§§P , 0.01, diabetic patients with HF vs. non-diabetic patients with HF.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 3 Serum levels of high-mobility group box-1 and other biological analytes in diabetic and non-diabetic patients according to different heart failure aetiology

Ischaemic cardiomyopathy (ICM) Dilated cardiomyopathy (DCM) Diabetes Aetiology Diabetes 3 aetiology

No diabetes (n 5 68) Diabetes (n 5 81) No diabetes (n 5 154) Diabetes (n 5 44) P1-value P2-value P3-value

HMGB1 (ng/mL) 5.31+4.87 5.60+7.94 6.75+8.48 6.87+8.92 0.147 0.066 0.431

cRAGE (pg/mL) 752.5+804.6 946.3+1102.5* 1001.3+1196.1} 1743.8+1838.0**,‡, §§ 0.015 0.003 0.072

esRAGE (pg/mL) 194.3+114.6 183.9+123.0 286.2+188.4} 241.2+294.1§ 0.078 0.015 0.248

hsCRP (mg/L) 10.68+15.74 17.19+25.52 20.14+34.11 23.28+39.84 0.037 0.261 0.352

NT-proBNP (pg/mL) 1612.5+1824.8 2987.1+3376.9 3357.1+3032.6 3944.4+4204.0 0.162 0.382 0.309

Values are given as mean+ standard deviation. P1, P2, and P3 values stand for the contributions of diabetes, aetiology, and diabetes–aetiology interaction to the differences of biological analytes among the four groups by two-way analysis ofvariance, respectively.*P , 0.05.**P , 0.01, diabetic patients of different aetiology vs. non-diabetic patients of the same aetiology.‡P , 0.01, diabetic patients of DCM vs. non-diabetic patients of ICM.§P , 0.05.§§P , 0.01, diabetic patients of DCM vs. diabetic patients of ICM.}P , 0.05, non-diabetic patients of DCM vs. non-diabetic patients of ICM.

HM

GB1

andR

AG

Ein

heartfailure

445

CI 1.455–4.582; P ¼ 0.009), blood urea nitrogen (OR: 1.596, 95%:CI 1.162–2.193; P ¼ 0.004), and systolic blood pressure (OR:0.386, 95%: CI 0.194–0.771; P ¼ 0.007, for a 1-SD increase)were independent determinants for HF in non-diabetic subjects,while blood urea nitrogen (OR: 1.314, 95%: CI 1.068–1.613; P ¼0.012), systolic blood pressure (OR: 0.386, 95%: CI 0.194–0.771;P ¼ 0.007, for 1-SD increase), and creatinine (OR: 2.364, 95%: CI1.228–4.213; P ¼ 0.018, for a 1-SD increase) were independentrisk factors for HF in diabetic subjects. When NT-proBNP,HMGB1, and RAGE isoform measurements together with theabove risk factors were included in the multivariable analysis(Model II), HMGB1, esRAGE, cRAGE, and NT-proBNP resultedin being independently associated with the presence of HF inboth non-diabetic and diabetic patients (Table 5).

Association of biological analytes withmortalityDuring a 2-year follow-up in HF patients, non-survivors had higherserum HMGB1 levels than survivors (12.41+ 17.39 vs. 6.01+7.02 ng/mL; P ¼ 0.002), and patients with one or more hospitaliz-ation for HF had higher serum cRAGE and NT-proBNP levels thanthose without (1279.5+ 1405.1 vs. 840.7+1053.3 pg/mL and

4584.45+ 5726.94 vs. 1025.4+ 1501.3 pg/mL, for serum cRAGEand NT-proBNP levels, respectively; for both P , 0.001).

DiscussionAlthough AGEs and several RAGE-related proteins are suggested tobe closely involved in atherosclerotic vascular disease in both diabeticand non-diabetic patients, their relation to HF has been much lessexplored. Our study is the first to show that elevated serum levelsof HMGB1 and cRAGE, and decreased esRAGE levels, are associatedwith HF in both diabetic and non-diabetic patients.

High-mobility group box-1, an intracellular regulator of geneexpression and also a RAGE ligand, has been shown to contributeto inflammatory reactions, sepsis, atherosclerosis, and re-perfusioninjury.15,26 –28 However, the in vivo pathological effects of HMGB1remain controversial. Andrassy et al.16 observed that adminis-tration of HMGB1 led to a pro-inflammatory response andworsened cardiac function in mice subjected to ischaemia–re-perfusion injury, while HMGB1 antagonists significantlyreduced such injury. In contrast, the experiments by Takahashiet al.24 demonstrated a clear-cut attenuation of local inflammationand fibrosis in rats with myocardial infarction given intra-myocardial HMGB1 injection, with an accompanying improvement

Figure 2 Correlation of serum high-mobility group box-1 (HMGB1) levels (on a natural logarithmic scale) with left ventricular volumes andejection fraction (LVEF), and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels (on a logarithmic scale) in heart failure (HF) patients.LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume. Due to positively skewed distribution of left ventri-cular ejection fraction values in heart failure patients, the correlation of high-mobility group box-1 levels with left ventricular ejection fractionwas analysed by the Spearman’s method, but displayed as Pearson’s linear correlation to show the crude distributions of values.

L.J. Wang et al.446

of cardiac function. The existence of such beneficial effects ofHMGB1 is also supported by the experiments of Limana et al.22

and Kitahara et al 23; the former showed myocardial regenerationinduced by exogenous HMGB1 administration through measure-ments of cardiac c-kit+ cell proliferation and differentiation;22

and the latter showed improvements of angiogenesis, cardiac func-tion, and survival after myocardial infarction.23

Our results, obtained in the largest population studied thus far inHF, evidenced a positive relationship of HMGB1 with HF, consist-ent with deleterious effects of HMGB1. In our study, the HMGB1value paralleled the clinical severity and worse outcome of HFindependent of the presence of diabetes. The reasons for theabove-mentioned discrepancies in the effects of HMGB1 in thesetting of HF remain unclear at present, but the variety of exper-imental conditions used in the different studies probably contrib-ute to the differences in results. In our study, we recruited HFpatients with different aetiologies, whose disease course wasmostly longer than 6 months, which is at variance with the acuteeffects observed in well-controlled experimental models or celllines. In conditions of ischaemia–reperfusion, a diffuse myocardialinjury provoked by the abundant production of oxygen-derivedfree radicals is the main pathophysiological mechanism, while per-sistent myocardial ischaemia is a dominant stimulus in mice withmyocardial infarction.24 Infection-associated myocarditis, auto-immune inflammation, and mutation-induced loss/dysfunction ofmatrix protein primarily initiate the development of dilated cardio-myopathy.29 Thus, the final results may vary between studies,depending on the combined pathogenetic mechanisms in which

HMGB1 and multiple other factors are involved with differingroles. Of note, the in vivo concentrations and distribution ofHMGB1 are quite different in previously reported studies, contri-buting to the disparity in findings.

Similar to the reports of Koyama et al.,30 we observed an elevationof cRAGE levels in HF patients, which was more remarkable in dia-betes and in dilated cardiomyopathy. Since RAGE cleavage by metal-loproteinase can be induced by HMGB1 and other inflammatorycytokines,18 the increase in cRAGE levels may represent a distinctivepathophysiological correlate of severe inflammatory reactions inthese diseases, whereby HMGB1 might actually be the trigger forincreased cRAGE concentrations. While cRAGE levels mightbecome elevated in response to a variety of other stimuli, includingAGEs,12 levels of HMGB1 would be more specific for HF, indepen-dent of the presence or absence of diabetes. This would explain theinteraction of HF and diabetes in determining the levels of cRAGE,but not of HMGB1.

Endogenous secretory RAGE is an endogenous protein thoughtto neutralize AGEs and some inflammatory cytokines, and acts as aprotective, anti-atherogenic factor. In the present study, weobserved that esRAGE levels were significantly lower in non-diabetic patients with HF than in normal controls, similar tolevels found in diabetes. These results indicate that protectivesystems against AGEs and inflammatory cytokines may be severelyimpaired in HF, and such impairment seems to be more prominentin ischaemic HF, where esRAGE levels were lower.

Taken together, the present findings show that increased levelsof HMGB1 and cRAGE and decreased esRAGE levels are

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 4 Association of biological analytes with disease severity in heart failure patients

Variables correlated Total HF patients Non-diabetic patients with HF Diabetic patients with HF

Correlation coefficient P-value Correlation coefficient P-value Correlation coefficient P-value

HMGB1—NT-proBNP 0.378 ,0.001 0.347 ,0.001 0.290 0.002

HMGB1—LAD 0.176 0.020 0.128 0.023 0.213 0.012

HMGB1—LVEDV 0.134 0.026 0.153 0.021 0.124 0.031

HMGB1—LVESV 0.153 0.011 0.178 0.009 0.138 0.021

HMGB1—LVEFa 20.127 0.021 20.181 0.011 20.103 0.024

HMGB1—NYHA classb 0.085 0.131 0.074 0.291 0.184 0.039

cRAGE—NT-proBNP 0.451 ,0.001 0.446 ,0.001 0.477 0.001

cRAGE—LAD 0.221 ,0.001 0.110 0.116 0.360 ,0.001

cRAGE—LVEDV 0.096 0.076 0.093 0.182 0.328 ,0.001

cRAGE—LVESV 0.134 0.013 0.050 0.482 0.358 ,0.001

cRAGE—LVEFa 20.179 0.001 20.015 0.663 20.356 ,0.001

cRAGE—NYHA classb 0.402 ,0.001 0.567 ,0.001 0.326 ,0.001

esRAGE—NT-proBNP 20.227 ,0.001 20.303 ,0.001 20.132 0.124

esRAGE—LAD 20.135 0.011 20.055 0.213 20.259 0.003

esRAGE—LVEDV 20.065 0.396 20.017 0.423 20.172 0.134

esRAGE—LVESV 20.152 0.258 20.138 0.321 20.175 0.048

esRAGE—LVEFa 20.049 0.341 20.019 0.316 20.074 0.423

esRAGE—NYHA classb 20.223 ,0.001 20.161 0.011 20.292 0.001

LAD, left atrial diameter (mm); LVEDV, left ventricular end-diastolic volume (mL); LVESV, left ventricular end-systolic volume (mL).a,bCorrelation coefficients were calculated by the Spearman’s method (NYHA class being categorical or LVEF non-normally distributed); the others were calculated by thePearson’s method (both variables being continuous).

HMGB1 and RAGE in heart failure 447

associated with the development and the severity of HF. Levels ofcRAGE were particularly elevated in HF patients with diabetes ordilated cardiomyopathy, as well as in patients experiencing are-hospitalization for HF during the follow-up. Moreover,HMGB1 levels were significantly higher in non-survivors vs. survi-vors within patients with HF. These findings support the notionthat RAGE, RAGE variants, and their ligands are closely—buteach of them differentially—marking the development or severityof HF, and possibly, because of their biological actions, involvedin the pathogenetic mechanism of HF. Here, HMGB1 was relatedto the development and progression of HF in both diabetic andnon-diabetic subjects, while cRAGE and esRAGE possibly play aselective role in diabetes-related worsening of heart function. Inour study, lower enrolment or the loss to follow-up of moresevere cases with the worst prognosis may have contributed tothe low mortality observed compared with that reported in pre-vious trials and registries as well as epidemiological studies.

Study limitationsWe recognize a few limitations in our study. First, this study wasmainly cross-sectional with little significance of the follow-updata due to the low numbers of events accrued, thereby only

allowing us to detect associations. Due to the study design, predic-tions and causal inferences are impossible. Second, although differ-ences in magnitude were identified with the current number ofsubjects, and fulfilling the original hypotheses, the sample size inour study is still relatively small, and larger prospective studiesare now warranted to confirm both the association and the predic-tive role of HMGB1 and related proteins with HF. Third, despitebeing statistically significant, the magnitude of the associationsfound (the slope of the relationship in regression analyses) wassmall, and interactions of variables not measured here may haveoccurred; importantly, information on diastolic function was notavailable. Fourth, all HF patients studied were Chinese; thus, itremains uncertain whether these results are fully applicable toother ethnicities. However, the identification of a new marker,with additional possible pathogenetic relevance, has to be regardedas a novelty in the area.

ConclusionsThe present study demonstrates higher serum levels of HMGB1and cRAGE, and lower levels of esRAGE in diabetic and non-diabetic patients with HF, associated with the severity of changes

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 5 Multivariable stepwise logistic regression model for the presence of heart failure

Variables Non-diabetic subjects Diabetic subjects

OR (95% confidence interval) P-value OR (95% confidence interval) P-value

Univariable conditional logistic regression adjusting for conventional risk factors (Model I)

Female gender 0.639 (0.254–1.606) 0.341 0.712 (0.270–1.877) 0.493

Age [years (SD)] 0.862 (0.574–1.295) 0.474 1.289 (0.860–1.931) 0.219

Hypertension 2.877 (0.999–6.351) 0.050 1.621 (0.636–4.134) 0.312

Smoking 2.235 (1.455–4.582) 0.009 1.174 (0.445–3.094) 0.746

Systolic blood pressure [mmHg (SD)] 0.386 (0.194–0.771) 0.007 0.543 (0.305–0.966) 0.038

Diastolic blood pressure [mmHg (SD)] 1.503 (0.883–2.559) 0.133 1.054 (0.688–1.613) 0.811

Blood urea nitrogen (mmol/L) 1.596 (1.162–2.193) 0.004 1.344 (1.068–1.690) 0.012

Creatinine [mmol/L (SD)] 1.230 (0.512–2.956) 0.602 2.364 (1.228–4.213) 0.018

Uric acid [mmol/L (SD)] 3.861 (0.556–8.797) 0.272 1.191 (0.875–1.622) 0.266

Fasting glucose (mmol/L) 1.938 (1.039–3.616) 0.038 0.877 (0.571–1.097) 0.154

Glycated haemoglobin (%) 1.625 (1.124–1.989) 0.023 0.932 (0.418–4.135) 0.236

Triglyceride (mmol/L) 2.011 (0.962–4.201) 0.063 0.962 (0.696–1.328) 0.435

Total cholesterol (mmol/L) 0.893 (0.557–1.430) 0.540 0.740 (0.507–1.078) 0.117

Backward stepwise regression adjusting for independent conventional risk factors and all biomarkers (Model II)

Smoking 1.325 (0.643–1.681) 0.219 — —

Systolic blood pressure (mmHg, SD) 2.512 (0.816–5.752) 0.108 0.754 (0.561–1.014) 0.062

Blood urea nitrogen (mmol/L) 1.237 (0.842–1.816) 0.278 1.555 (1.001–2.417) 0.050

Creatinine [mmol/L (SD)] — — 1.429 (0.852–2.293) 0.176

Fasting glucose (mmol/L) 1.282 (0.926–1.775) 0.135 — —

Glycated haemoglobin (%) 1.425 (1.013–1.938) 0.011 — —

NT-proBNP [pg/mL (SD/2)] 1.512 (1.043–1.886) 0.001 1.602 (1.261–2.036) 0.001

HMGB1 [ng/mL (SD)] 1.681 (1.164–1.696) 0.002 1.357 (1.068–1.722) 0.008

cRAGE [pg/mL (SD/2)] 1.402 (1.055–2.069) 0.043 1.256 (1.150–1.971) 0.032

esRAGE [pg/mL (SD)] 0.441 (0.283–0.689) 0.008 0.394 (0.196–0.878) 0.023

OR, odd ratios.

L.J. Wang et al.448

in cardiac function, symptoms, and clinical outcome. Such associ-ations are now worth further exploration.

FundingThis study was supported by a key grant from the Science and Tech-nology Commission of Shanghai Municipality-‘Optimal Therapy ofMyocardial Infarction with Diabetes’ (05DZ19503).

Conflict of interest: none declared.

References1. Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end pro-

ducts: sparking the development of diabetic vascular injury. Circulation 2006;114:597–605.

2. Kilhovd BK, Berg TJ, Birkeland KI, Thorsby P, Hanssen KF. Serum levels ofadvanced glycation end products are increased in patients with type 2 diabetesand coronary heart disease. Diabetes Care 1999;22:1543–1548.

3. Kiuchi K, Nejima J, Takano T, Ohta M, Hashimoto H. Increased serum concen-trations of advanced glycation end products: a marker of coronary arterydisease activity in type 2 diabetic patients. Heart 2001;85:87–91.

4. Tan KC, Chow WS, Ai VH, Metz C, Bucala R, Lam KS. Advanced glycation endproducts and endothelial dysfunction in type 2 diabetes. Diabetes Care 2002;25:1055–1059.

5. Kanauchi M, Tsujimoto N, Hashimoto T. Advanced glycation end products innondiabetic patients with coronary artery disease. Diabetes Care 2001;24:1620–1623.

6. Avendano GF, Agarwal RK, Bashey RI, Lyons MM, Soni BJ, Jyothirmayi GN,Regan TJ. Effects of glucose intolerance on myocardial function and collagen-linked glycation. Diabetes 1999;48:1443–1447.

7. Asif M, Egan J, Vasan S, Jyothirmayi GN, Masurekar MR, Lopez S, Williams C,Torres RL, Wagle D, Ulrich P, Cerami A, Brines M, Regan TJ. An advanced glyca-tion endproduct cross-link breaker can reverse age-related increases in myocar-dial stiffness. Proc Natl Acad Sci USA 2000;97:2809–2813.

8. Aronson D. Cross-linking of glycated collagen in the pathogenesis of arterial andmyocardial stiffening of aging and diabetes. J Hypertens 2003;21:3–12.

9. Hartog JW, van de Wal RM, Schalkwijk CG, Miyata T, Jaarsma W, Plokker HW,van Wijk LM, Smit AJ, van Veldhuisen DJ, Voors AA. Advanced glycation end-products, anti-hypertensive treatment and diastolic function in patients withhypertension and diastolic dysfunction. Eur J Heart Fail 2010;12:397–403.

10. Hartog JW, Voors AA, Schalkwijk CG, Scheijen J, Smilde TD, Damman K,Bakker SJ, Smit AJ, van Veldhuisen DJ. Clinical and prognostic value of advancedglycation end-products in chronic heart failure. Eur Heart J 2007;28:2879–2885.

11. Koyama Y, Takeishi Y, Arimoto T, Niizeki T, Shishido T, Takahashi H, Nozaki N,Hirono O, Tsunoda Y, Nitobe J, Watanabe T, Kubota I. High serum level of pen-tosidine, an advanced glycation end product (AGE), is a risk factor of patients withheart failure. J Card Fail 2007;13:199–206.

12. Basta G. Receptor for advanced glycation endproducts and atherosclerosis: frombasic mechanisms to clinical implications. Atherosclerosis 2008;196:9–21.

13. Lu L, Pu LJ, Zhang Q, Wang LJ, Kang S, Zhang RY, Chen QJ, Wang JG, DeCaterina R, Shen WF. Increased glycated albumin and decreased esRAGE levelsare related to angiographic severity and extent of coronary artery disease inpatients with type 2 diabetes. Atherosclerosis 2009;206:540–545.

14. Chen Y, Pat B, Zheng J, Cain L, Powell P, Shi K, Sabri A, Husain A, Dell’italia LJ.Tumor necrosis factor-alpha produced in cardiomyocytes mediates a predomi-nant myocardial inflammatory response to stretch in early volume overload.J Mol Cell Cardiol 2010;49:70–78.

15. Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necroticcells triggers inflammation. Nature 2002;418:191–195.

16. Andrassy M, Volz HC, Igwe JC, Funke B, Eichberger SN, Kaya Z, Buss S,Autschbach F, Pleger ST, Lukic IK, Bea F, Hardt SE, Humpert PM, Bianchi ME,Mairbaurl H, Nawroth PP, Remppis A, Katus HA, Bierhaus A. High-mobilitygroup box-1 in ischemia-reperfusion injury of the heart. Circulation 2008;117:3216–3226.

17. Sparvero LJ, Asafu-Adjei D, Kang R, Tang D, Amin N, Im J, Rutledge R, Lin B,Amoscato AA, Zeh HJ, Lotze MT. RAGE (Receptor for Advanced Glycation End-products), RAGE ligands, and their role in cancer and inflammation. J Transl Med2009;7:17.

18. Raucci A, Cugusi S, Antonelli A, Barabino SM, Monti L, Bierhaus A, Reiss K,Saftig P, Bianchi ME. A soluble form of the receptor for advanced glycation end-products (RAGE) is produced by proteolytic cleavage of the membrane-boundform by the sheddase a disintegrin and metalloprotease 10 (ADAM10). FASEB J2008;22:3716–3727.

19. Yan XX, Lu L, Peng WH, Wang LJ, Zhang Q, Zhang RY, Chen QJ, Shen WF.Increased serum HMGB1 level is associated with coronary artery disease in non-diabetic and type 2 diabetic patients. Atherosclerosis 2009;205:544–548.

20. Kohno T, Anzai T, Naito K, Miyasho T, Okamoto M, Yokota H, Yamada S,Maekawa Y, Takahashi T, Yoshikawa T, Ishizaka A, Ogawa S. Role of high-mobilitygroup box 1 protein in post-infarction healing process and left ventricular remo-delling. Cardiovasc Res 2009;81:565–573.

21. Palumbo R, Sampaolesi M, De Marchis F, Tonlorenzi R, Colombetti S, Mondino A,Cossu G, Bianchi ME. Extracellular HMGB1, a signal of tissue damage, inducesmesoangioblast migration and proliferation. J Cell Biol 2004;164:441–449.

22. Limana F, Germani A, Zacheo A, Kajstura J, Di Carlo A, Borsellino G, Leoni O,Palumbo R, Battistini L, Rastaldo R, Muller S, Pompilio G, Anversa P,Bianchi ME, Capogrossi MC. Exogenous high-mobility group box 1 proteininduces myocardial regeneration after infarction via enhanced cardiac C-kit+cell proliferation and differentiation. Circ Res 2005;97:e73–e83.

23. Kitahara T, Takeishi Y, Harada M, Niizeki T, Suzuki S, Sasaki T, Ishino M, Bilim O,Nakajima O, Kubota I. High-mobility group box 1 restores cardiac function aftermyocardial infarction in transgenic mice. Cardiovasc Res 2008;80:40–46.

24. Takahashi K, Fukushima S, Yamahara K, Yashiro K, Shintani Y, Coppen SR,Salem HK, Brouilette SW, Yacoub MH, Suzuki K. Modulated inflammation byinjection of high-mobility group box 1 recovers post-infarction chronicallyfailing heart. Circulation 2008;118(14 Suppl.):S106–S114.

25. Heymans S, Hirsch E, Anker SD, Aukrust P, Balligand JL, Cohen-Tervaert JW,Drexler H, Filippatos G, Felix SB, Gullestad L, Hilfiker-Kleiner D, Janssens S,Latini R, Neubauer G, Paulus WJ, Pieske B, Ponikowski P, Schroen B,Schultheiss HP, Tschope C, Van Bilsen M, Zannad F, McMurray J, Shah AM. Inflam-mation as a therapeutic target in heart failure? A scientific statement from theTranslational Research Committee of the Heart Failure Association of the Euro-pean Society of Cardiology. Eur J Heart Fail 2009;11:119–129.

26. Andersson U, Wang H, Palmblad K, Aveberger AC, Bloom O,Erlandsson-Harris H, Janson A, Kokkola R, Zhang M, Yang H, Tracey KJ. Highmobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesisin human monocytes. J Exp Med 2000;192:565–570.

27. Kalinina N, Agrotis A, Antropova Y, DiVitto G, Kanellakis P, Kostolias G,Ilyinskaya O, Tararak E, Bobik A. Increased expression of the DNA-binding cyto-kine HMGB1 in human atherosclerotic lesions: role of activated macrophages andcytokines. Arterioscler Thromb Vasc Biol 2004;24:2320–2325.

28. Porto A, Palumbo R, Pieroni M, Aprigliano G, Chiesa R, Sanvito F, Maseri A,Bianchi ME. Smooth muscle cells in human atherosclerotic plaques secrete andproliferate in response to high mobility group box 1 protein. FASEB J 2006;20:2565–2566.

29. Maisch B, Richter A, Sandmoller A, Portig I, Pankuweit S. Inflammatory dilatedcardiomyopathy (DCMI). Herz 2005;30:535–544.

30. Koyama Y, Takeishi Y, Niizeki T, Suzuki S, Kitahara T, Sasaki T, Kubota I. Solublereceptor for advanced glycation end products (RAGE) is a prognostic factor forheart failure. J Card Fail 2008;14:133–139.

HMGB1 and RAGE in heart failure 449