These authors contributed equally to this manuscript.
Increased serum high-mobility group box-1 and cleaved receptor for advanced glycation endproducts levels and decreased endogenous secretory receptor for advanced glycation endproducts levels in diabetic and non-diabetic patients with heart failure
Version of Record online: 18 FEB 2014
Published on behalf of the European Society of Cardiology. All rights reserved. © 2011 the Authors
European Journal of Heart Failure
Volume 13, Issue 4, pages 440–449, April 2011
How to Cite
Wang, L. J., Lu, L., Zhang, F. R., Chen, Q. J., De Caterina, R. and Shen, W. F. (2011), Increased serum high-mobility group box-1 and cleaved receptor for advanced glycation endproducts levels and decreased endogenous secretory receptor for advanced glycation endproducts levels in diabetic and non-diabetic patients with heart failure. European Journal of Heart Failure, 13: 440–449. doi: 10.1093/eurjhf/hfq231
- Issue online: 18 FEB 2014
- Version of Record online: 18 FEB 2014
- Manuscript Accepted: 30 SEP 2010
- Manuscript Revised: 25 SEP 2010
- Manuscript Received: 25 APR 2010
- Chronic heart failure;
High-mobility group box-1 (HMGB1) is a ligand for the receptor for advanced glycation endproducts (RAGE). An HMGB1–RAGE interaction has been implicated in cardiac dysfunction. We assessed the association of HMGB1 and RAGE isoforms with heart failure (HF) in diabetic and non-diabetic patients.
Methods and results
We assayed serum levels of HMGB1, cleaved RAGE (cRAGE), endogenous secretory RAGE (esRAGE), high-sensitivity C-reactive protein (hsCRP), and N-terminal pro-brain natriuretic peptide (NT-proBNP) in parallel with assessment 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 levels lower in HF patients than in subjects without HF (for all; P < 0.01), with higher levels of cRAGE and hsCRP in diabetic HF vs. non-diabetic HF patients (P < 0.01). For HF patients—with or without diabetes—HMGB1 levels correlated positively with left ventricular end-diastolic and end-systolic volumes (r = 0.267 and r = 0.321, respectively) and NT-proBNP values (r = 0.497), and were inversely related to ejection fraction (r = −0.461; all P < 0.001). Serum cRAGE levels correlated with NT-proBNP values (r = 0.451) and New York Heart Association functional class (r = 0.402; both P < 0.001). Multivariable regression analysis revealed that HMGB1, cRAGE, and esRAGE were consistently associated with HF in diabetic and non-diabetic patients.
Heart failure patients have increased serum HMGB1 and cRAGE and decreased esRAGE levels, and these are related to the severity of HF in both diabetic and non-diabetic patients. Such associations are worth further investigation.
Interaction of advanced glycation endproducts (AGEs) with the main receptor (RAGE), and subsequent intracellular signalling, has been implicated in diabetic complications.1 Advanced glycation endproducts, acting as pro-inflammatory triggers, are associated with endothelial dysfunction and coronary artery disease, in both diabetic 2–4 and non-diabetic patients.5 Interestingly, AGEs have also been recently implicated in cardiac dysfunction,6–9 thus emerging as a risk factor for poor clinical outcomes in chronic heart failure (HF).10,11
Besides full-length membrane-bound RAGE, two major additional isoforms have been recently identified, namely the endogenous secretory RAGE (esRAGE) and a cleaved form of RAGE (cRAGE).12 Endogenous secretory RAGE, a quantitatively minor isoform, is generated through alternative splicing of pre-mRNA; cRAGE is proteolytically cleaved from the cell surface by matrix metalloproteinases, and then shed into the bloodstream. Both variants may act as decoy ligands for AGEs and several inflammatory cytokines. Recent studies have, however, suggested a reciprocal relationship between these two ligands, whereby decreased esRAGE and/or increased cRAGE levels are biomarkers of heightened ligand–RAGE interaction in diabetes, atherosclerosis, and other inflammatory diseases, possibly underscoring an inadequate endogenous protective response.13 This suggestion has been recently extended to HF, where esRAGE levels are considered a negative prognostic factor.14
High-mobility group box-1 (HMGB1) is a nuclear DNA-binding protein, passively released from necrotic cells and actively secreted by activated immune cells.15 Recently, HMGB1 has been demonstrated to be a novel ligand for RAGE, cRAGE, and esRAGE.16,17 The engagement of HMGB1 with RAGE promotes the shedding of the receptor.18 Its signalling via RAGE activates inflammatory pathways and intensifies cellular oxidative stress, which results in profuse production of inflammatory cytokines and elevated expression of adhesion molecules.17 Increased HMGB1 levels are associated with ischaemia–reperfusion injury in mice, and with coronary artery disease in diabetic patients,16,19 and are also involved in post-infarction inflammatory response and left ventricular remodelling.20 On the other hand, low levels of HMGB1 have also distinctly favourable biological properties, being capable of attracting stem cells,21 facilitating myocardial cell regeneration and differentiation,22 enhancing angiogenesis, and consequently improving myocardial function and survival after myocardial infarction.23 Indeed, low-grade inflammation elicited by intra-myocardial injection 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 clues about 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 progression of HF.25 Based on these findings, we hypothesized that HMGB1 concentrations may mark—and perhaps contribute to—the development of HF, with accompanying esRAGE and cRAGE changes. We predicted a graded relationship between HF severity and changes in plasma levels of HMGB1 and related proteins. We therefore evaluated the association of serum concentrations of HMGB1 (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 precursor of brain natriuretic peptide (NT-proBNP) (secondary objectives) as a HF biomarker, in non-diabetic and type 2 diabetic patients with chronic HF.
The study protocol was approved by the local hospital Ethics Committee, and written informed consent was obtained from all participants.
We restricted the inclusion to Chinese patients of Han nationality with systolic chronic HF due to ischaemic or idiopathic dilated cardiomyopathy. The study, therefore, included 149 consecutive patients with ischaemic 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 cardiomyopathy (mean age: 57.0 ± 13.5 years; duration of disease: 2.9 ± 3.3 years) recruited between January 2000 and May 2006 from three teaching hospitals affiliated to the Shanghai Jiaotong University School of Medicine in China. Systolic HF was diagnosed according to the European Society of Cardiology guidelines, including patients with symptoms or signs of HF and left ventricular ejection fraction (LVEF) <45%, assessed by echocardiography. For patients with idiopathic dilated cardiomyopathy and cardiovascular risk factors (e.g. with associated type 2 diabetes), the presence of significant coronary artery disease was excluded by coronary angiography. Of the 347 patients with HF, 222 were non-diabetic and 125 had type 2 diabetes. Diabetes was defined according to the American Diabetes Association criteria as: two fasting plasma glucose levels ≥7.0 mmol/L, or symptoms of diabetes plus a casual post-prandial plasma glucose reading ≥11.1 mmol/L, or a 2 h glucose reading ≥11.1 mmol/L after a 75 g glucose load, or taking oral hypoglycaemic drugs or parenteral insulin. To avoid confounding variables, we excluded patients with a history of viral myocarditis, hypertrophic cardiomyopathy, primary valvular disease, or pulmonary heart disease. We also excluded patients with type 1 diabetes, chronic viral or bacterial infections, tumours, or immune disorders. Detailed information was obtained on demographics, clinical manifestation, and medications, as well as New York Heart Association (NYHA) functional class, and echocardiographic measurements were used to evaluate HF severity.
Two hundred and twenty normal Chinese subjects (132 men and 88 women; mean age: 60 ± 13 years) and 79 Chinese patients with type 2 diabetes only (37 men and 42 women; mean age: 64 ± 10 years) served as normal and diabetic controls, respectively. Their gender distribution was the same as in the general population; age was purposely matched between controls and the HF population. Detailed medical and family history was taken, and fasting blood samples were collected during an annual physical check-up. In the normal controls, serum levels of glucose, lipid profiles, liver and renal function tests, and the electrocardiogram were normal in all subjects, and none had a history of cardiovascular diseases (including past history of angina/myocardial infarction). In the diabetic control group, we excluded patients with prior coronary heart disease, stroke, or severe renal dysfunction.
Peripheral venous blood samples were collected after an overnight fast. Serum glucose, blood urea nitrogen, creatinine, uric acid, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, lipoprotein (a), apolipoprotein A, apolipoprotein B, and triglycerides were measured using standard laboratory techniques on a Hitachi 912 Analyser (Roche Diagnostics, Mannheim, Germany). Serum NT-proBNP was determined using a commercially available electrochemiluminescence immunoassay kit (Roche Diagnostics). Serum HMGB1 levels were assessed with an enzyme-linked immunosorbent assay (ELISA) kit (HMGB1 ELISA kit II; Shino-Test Corporation, Tokyo, Japan) according to the manufacturer's instructions. The detection limit for HMGB1 was 0.2 ng/mL, with an inter-assay coefficient of variation <10%. Levels of cRAGE and esRAGE were also measured using ELISA kits (Quantikine; R&D Systems, MN, USA; B-Bridge International, Mountain View, CA, USA, respectively).
Transthoracic two-dimensional echocardiography was performed using a Hewlett-Packard Sonos 2500 (Hewlett-Packard, San Diego, CA, USA) or a GE Vivid-7 system (General Electric Vingmed Sound AS, Horton, Norway) equipped with 2.5 or 1.7/3.4 MHz transducers, respectively. Images were obtained at rest with the patient lying in the left lateral decubitus position at end-expiration. Left ventricular end-diastolic and end-systolic volumes were measured according to the biplane Simpson's method based on the American Society of Echocardiography recommendations, and LVEF was calculated. An average of three consecutive cardiac cycles was used for each patient.
All HF patients were prescribed standard HF treatments and were seen every 1–3 months in a dedicated HF clinic. During each visit, heart rate, blood pressure, and new clinical manifestations were recorded; echocardiography was performed every 6 months. Adverse events (hospitalization or death from HF) were recorded during each visit or by telephone with patients or family members. Hospitalization for HF was defined as that due to progressive fluid retention and the need to increase or change medications. Two trained physicians independently reviewed all medical notes, including forms from visits to the emergency department and hospital medical records.
Continuous 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 hoc between-group analysis or by the non-parametric Kruskal–Wallis test. Categorical data were summarized as frequencies or percentages, and differences between groups were evaluated by the χ2 test. The sample size (347 patients with HF and 299 subjects without HF, with or without diabetes) was such to yield >80% power to detect differences between HF and non-HF patients for HMGB1, NT-proBNP, cRAGE, and hsCRP (for all, 99% power both in non-diabetic and diabetic patients), as well as esRAGE levels (99% in non-diabetic and 87% in diabetic patients, respectively) under a type I error probability of 0.05 (1−α) for a two-sided test.
Correlations of serum HMGB1 (on a logarithmic scale) with other biomarkers or echocardiographic measurements (left atrial diameters and ventricular volumes) were assessed by Pearson's test, and associations of these biomarkers with LVEF (not normally distributed) or NYHA functional class (categorical variable) by Spearman's test. The linear correlation of LVEF with HMGB1 was still represented with the Pearson's method to represent their crude distributions. We used two models in multivariable logistic regression analysis for the presence of HF in non-diabetic and diabetic subjects, respectively. In Model I, multivariable adjustment was made for conventional risk factors measured at baseline examination, and included age, gender, smoking, hypertension, systolic/diastolic blood pressure, triglycerides, total cholesterol, blood urea nitrogen, creatinine, uric acid, fasting glucose, and glycated haemoglobin (by a conditional logistic regression method). In Model II, the multivariable-adjusted odd ratios (ORs) and their 95% conﬁdence intervals (CI) for HF associated with the biomarkers of interest were compared with the respective normal- and diabetic-matched controls, synchronously estimated together with significantly 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 software (SPSS, Inc., Chicago, IL, USA).
The diabetic and non-diabetic patients with HF were more frequently male and cigarette smokers than the normal or diabetic controls. Compared with non-diabetic patients with HF, those with both HF and diabetes were older and had a higher ratio of ischaemic vs. non-ischaemic aetiology, a poorer renal function, and more abnormal lipid profiles. No significant differences between HF patients with and without diabetes were observed for left ventricular end-diastolic or end-systolic volumes or LVEF, with the exception of a larger left atrial size in diabetic patients with HF. During a 2-year follow-up, 22 out of 125 (17.6%) diabetic and 23 out of 222 (10.4%) non-diabetic patients with HF died of sudden cardiac death or refractory HF (Table 1).
|Non-diabetic patients||Diabetic patients|
|No HF (n = 220)||HF (n = 222)||P-value||No HF (n = 79)||HF (n = 125)||P-value|
|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 (μmol/L)||79.1 (17.3)||104.6 (70.5)||<0.001||84.2 (21.9)||104.8 (42.5)‡||<0.001|
|Uric acid (μmol/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|
|β-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|
Influence of heart failure and its aetiology on biological analytes
Serum 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 ± 17.08 vs. 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 population of patients with HF than in those without HF (all P < 0.01), while elevated levels of cRAGE (643.5 ± 1176.3 vs. 594.5 ± 401.5 pg/mL in diabetic and non-diabetic subjects, respectively), hsCRP (11.39 ± 9.31 vs. 5.15 ± 4.38 mg/L; both P < 0.05), and reduced esRAGE levels (253.5 ± 109.9 vs. 415.3 ± 131.3 pg/mL; P < 0.001) were present in association with diabetes in patients without HF. In other words, levels of HMGB1 and NT-proBNP, in contrast to the other analytes assayed, were selectively influenced by the presence of HF. Higher levels of cRAGE and hsCRP, but not of HMGB1 or esRAGE, were observed in diabetic vs. non-diabetic patients with HF (for all; P < 0.01; Figure 1 and Table 2). In addition, esRAGE levels were selectively related to the aetiology of HF, with higher levels in dilated vs. ischaemic cardiomyopathy (260.5 ± 290.3 vs. 222.3 ± 182.0 pg/mL; P = 0.015); whereas cRAGE levels were influenced by both the aetiology of HF (P = 0.015) and diabetes (P = 0.003), with the highest levels seen in diabetic patients with dilated cardiomyopathy (1743.8 ± 1838.0 pg/mL), and the lowest levels in non-diabetic patients with ischaemic cardiomyopathy (752.5 ± 804.6 pg/mL). However, HMGB1 levels were not influenced by aetiology (5.38 ± 0.69 vs. 6.91 ± 0.72 ng/mL in ischaemic and dilated cardiomyopathy, respectively; P = 0.066) or the presence of diabetes (6.14 ± 0.79 vs. 6.10 ± 0.60 ng/mL in diabetic and non-diabetic patients, respectively; P = 0.123) in HF (Table 3).
|Non-diabetic patients||Diabetic patients||Diabetes||HF||Diabetes × HF|
|No HF (n = 220)||HF (n = 222)||P-value||No HF (n = 79)||HF (n = 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|
|Ischaemic cardiomyopathy (ICM)||Dilated cardiomyopathy (DCM)||Diabetes||Aetiology||Diabetes × aetiology|
|No diabetes (n = 68)||Diabetes (n = 81)||No diabetes (n = 154)||Diabetes (n = 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|
Serum HMGB1 levels correlated positively with hsCRP (Pearson's r = 0.208; P < 0.001) and cRAGE values (Pearson's r = 0.086; P = 0.042), and were inversely related to esRAGE (Pearson's r = −0.203; P < 0.001). Serum esRAGE correlated negatively with hsCRP levels (Pearson's r = −0.249; P < 0.001).
Association of biological analytes with heart failure severity
The relationship between serum measurements of HMGB1 levels and HF severity is depicted in Figure 2. High-mobility group box-1 was positively related to NT-proBNP (Pearson's r = 0.497; P < 0.001), and left ventricular end-diastolic and end-systolic volumes (Pearson's r = 0.267 and r = 0.321, respectively; both P < 0.001), but was inversely related to LVEF (Spearman's r = −0.461; P < 0.001) in both diabetic and non-diabetic HF patients. Moreover, HMGB1 levels showed a positive correlation with NYHA functional class in HF diabetic patients (Spearman's r = 0.184; P = 0.039), but not in HF non-diabetic patients (Spearman's r = 0.074; P = 0.291).
Similarly, serum levels of cRAGE were positively related to NT-proBNP levels independent of diabetes (Spearman's r = 0.451; P < 0.001), but were positively associated with left atrial diameters (Pearson's r = 0.360; P < 0.001), left ventricular end-diastolic and end-systolic volumes (Pearson's r = 0.358 and r = 0.328, respectively; both P < 0.001), and inversely related to LVEF (Spearman's r = −0.356; P < 0.001) only in diabetic patients with HF. In addition, in both non-diabetic and diabetic patients with HF, serum cRAGE levels correlated significantly with NYHA functional class (Spearman's r = 0.326 and r = 0.567, respectively; both P < 0.001). There was a stepwise elevation of cRAGE levels with worsening NYHA functional classes [median and 25–75% percentiles: 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 with NT-proBNP levels (Pearson's r = −0.303; P < 0.001) only in non-diabetic patients, and with left atrial diameters (Pearson's r = −0.259; P = 0.003) and left ventricular end-systolic volumes (Pearson's r = −0.175; P = 0.048) only in diabetic patients with HF. No significant correlation between esRAGE and LVEF was found in either group (both P > 0.05). In addition, esRAGE levels were negatively related to NYHA functional class in diabetic and non-diabetic patients with HF (Spearman's r = −0.292 (P = 0.001) and r = −0.161 (P = 0.011), respectively). The detailed association between these biological analytes and HF severity is shown in Table 4.
|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|
Multivariable logistic regression analysis for the presence of heart failure
Multivariable stepwise logistic regression analysis run in all subjects, with or without HF, revealed that, adjusting for traditional cardiovascular risk factors (Model I), smoking (OR: 2.235, 95%: CI 1.455–4.582; P = 0.009), blood urea nitrogen (OR: 1.596, 95%: CI 1.162–2.193; P = 0.004), and systolic blood pressure (OR: 0.386, 95%: CI 0.194–0.771; P = 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 = 0.012), systolic blood pressure (OR: 0.386, 95%: CI 0.194–0.771; P = 0.007, for 1-SD increase), and creatinine (OR: 2.364, 95%: CI 1.228–4.213; P = 0.018, for a 1-SD increase) were independent risk factors for HF in diabetic subjects. When NT-proBNP, HMGB1, and RAGE isoform measurements together with the above risk factors were included in the multivariable analysis (Model II), HMGB1, esRAGE, cRAGE, and NT-proBNP resulted in being independently associated with the presence of HF in both non-diabetic and diabetic patients (Table 5).
|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 [µmol/L (SD)]||1.230 (0.512–2.956)||0.602||2.364 (1.228–4.213)||0.018|
|Uric acid [µmol/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)|
|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 [µmol/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|
Association of biological analytes with mortality
During a 2-year follow-up in HF patients, non-survivors had higher serum HMGB1 levels than survivors (12.41 ± 17.39 vs. 6.01 ± 7.02 ng/mL; P = 0.002), and patients with one or more hospitalization for HF had higher serum cRAGE and NT-proBNP levels than those 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 cRAGE and NT-proBNP levels, respectively; for both P < 0.001).
Although AGEs and several RAGE-related proteins are suggested to be closely involved in atherosclerotic vascular disease in both diabetic and non-diabetic patients, their relation to HF has been much less explored. Our study is the first to show that elevated serum levels of HMGB1 and cRAGE, and decreased esRAGE levels, are associated with HF in both diabetic and non-diabetic patients.
High-mobility group box-1, an intracellular regulator of gene expression and also a RAGE ligand, has been shown to contribute to inflammatory reactions, sepsis, atherosclerosis, and re-perfusion injury.15,26–28 However, the in vivo pathological effects of HMGB1 remain controversial. Andrassy et al.16 observed that administration of HMGB1 led to a pro-inflammatory response and worsened cardiac function in mice subjected to ischaemia–re-perfusion injury, while HMGB1 antagonists significantly reduced such injury. In contrast, the experiments by Takahashi et al.24 demonstrated a clear-cut attenuation of local inflammation and fibrosis in rats with myocardial infarction given intra-myocardial HMGB1 injection, with an accompanying improvement of cardiac function. The existence of such beneficial effects of HMGB1 is also supported by the experiments of Limana et al.22 and Kitahara et al 23; the former showed myocardial regeneration induced by exogenous HMGB1 administration through measurements of cardiac c-kit+ cell proliferation and differentiation;22 and the latter showed improvements of angiogenesis, cardiac function, and survival after myocardial infarction.23
Our results, obtained in the largest population studied thus far in HF, evidenced a positive relationship of HMGB1 with HF, consistent with deleterious effects of HMGB1. In our study, the HMGB1 value paralleled the clinical severity and worse outcome of HF independent of the presence of diabetes. The reasons for the above-mentioned discrepancies in the effects of HMGB1 in the setting of HF remain unclear at present, but the variety of experimental conditions used in the different studies probably contribute to the differences in results. In our study, we recruited HF patients with different aetiologies, whose disease course was mostly longer than 6 months, which is at variance with the acute effects observed in well-controlled experimental models or cell lines. In conditions of ischaemia–reperfusion, a diffuse myocardial injury provoked by the abundant production of oxygen-derived free radicals is the main pathophysiological mechanism, while persistent myocardial ischaemia is a dominant stimulus in mice with myocardial infarction.24 Infection-associated myocarditis, autoimmune inflammation, and mutation-induced loss/dysfunction of matrix protein primarily initiate the development of dilated cardiomyopathy.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 differing roles. Of note, the in vivo concentrations and distribution of HMGB1 are quite different in previously reported studies, contributing to the disparity in findings.
Similar to the reports of Koyama et al.,30 we observed an elevation of cRAGE levels in HF patients, which was more remarkable in diabetes and in dilated cardiomyopathy. Since RAGE cleavage by metalloproteinase can be induced by HMGB1 and other inflammatory cytokines,18 the increase in cRAGE levels may represent a distinctive pathophysiological correlate of severe inflammatory reactions in these diseases, whereby HMGB1 might actually be the trigger for increased cRAGE concentrations. While cRAGE levels might become elevated in response to a variety of other stimuli, including AGEs,12 levels of HMGB1 would be more specific for HF, independent of the presence or absence of diabetes. This would explain the interaction of HF and diabetes in determining the levels of cRAGE, but not of HMGB1.
Endogenous secretory RAGE is an endogenous protein thought to neutralize AGEs and some inflammatory cytokines, and acts as a protective, anti-atherogenic factor. In the present study, we observed that esRAGE levels were significantly lower in non-diabetic patients with HF than in normal controls, similar to levels found in diabetes. These results indicate that protective systems against AGEs and inflammatory cytokines may be severely impaired in HF, and such impairment seems to be more prominent in ischaemic HF, where esRAGE levels were lower.
Taken together, the present findings show that increased levels of HMGB1 and cRAGE and decreased esRAGE levels are associated with the development and the severity of HF. Levels of cRAGE were particularly elevated in HF patients with diabetes or dilated cardiomyopathy, as well as in patients experiencing a re-hospitalization for HF during the follow-up. Moreover, HMGB1 levels were significantly higher in non-survivors vs. survivors within patients with HF. These findings support the notion that RAGE, RAGE variants, and their ligands are closely—but each of them differentially—marking the development or severity of HF, and possibly, because of their biological actions, involved in the pathogenetic mechanism of HF. Here, HMGB1 was related to the development and progression of HF in both diabetic and non-diabetic subjects, while cRAGE and esRAGE possibly play a selective role in diabetes-related worsening of heart function. In our study, lower enrolment or the loss to follow-up of more severe cases with the worst prognosis may have contributed to the low mortality observed compared with that reported in previous trials and registries as well as epidemiological studies.
We recognize a few limitations in our study. First, this study was mainly cross-sectional with little significance of the follow-up data due to the low numbers of events accrued, thereby only allowing us to detect associations. Due to the study design, predictions and causal inferences are impossible. Second, although differences in magnitude were identified with the current number of subjects, and fulfilling the original hypotheses, the sample size in our study is still relatively small, and larger prospective studies are now warranted to confirm both the association and the predictive role of HMGB1 and related proteins with HF. Third, despite being statistically significant, the magnitude of the associations found (the slope of the relationship in regression analyses) was small, and interactions of variables not measured here may have occurred; importantly, information on diastolic function was not available. Fourth, all HF patients studied were Chinese; thus, it remains uncertain whether these results are fully applicable to other ethnicities. However, the identification of a new marker, with additional possible pathogenetic relevance, has to be regarded as a novelty in the area.
The present study demonstrates higher serum levels of HMGB1 and cRAGE, and lower levels of esRAGE in diabetic and non-diabetic patients with HF, associated with the severity of changes in cardiac function, symptoms, and clinical outcome. Such associations are now worth further exploration.
This study was supported by a key grant from the Science and Technology Commission of Shanghai Municipality-‘Optimal Therapy of Myocardial Infarction with Diabetes' (05DZ19503).
Conflict of interest: none declared.
- 25Inflammation as a therapeutic target in heart failure? A scientific statement from the Translational Research Committee of the Heart Failure Association of the European Society of Cardiology Eur J Heart Fail 2009 11 119 129