Clinical Biochemistry 48 (2015) 297–301
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Dynamic changes in sRAGE levels and relationship with cardiac function in STEMI patients Louise J.N. Jensen a,b,⁎, Søren Lindberg c, Søren Hoffmann c, Allan Z. Iversen c, Sune H. Pedersen c, Rasmus Møgelvang c, Søren Galatius c, Allan Flyvbjerg a,b, Jan S. Jensen c,d, Mette Bjerre a,b a
The Medical Research Laboratory, Department of Clinical Medicine, Aarhus University, Noerrebrogade 44, DK-8000 Aarhus C, Denmark Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Noerrebrogade 44, DK-8000 Aarhus C, Denmark Department of Cardiology P, Gentofte University Hospital, Niels Andersens Vej 65, DK-2900 Hellerup, Denmark d Institute of Clinical Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen N, Denmark b c
a r t i c l e
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Article history: Received 29 August 2014 Received in revised form 19 November 2014 Accepted 23 December 2014 Available online 3 January 2015 Keywords: Soluble receptor of advanced glycation end-products sRAGE Biomarker Acute myocardial infarction Primary percutaneous coronary intervention Left ventricular ejection fraction
a b s t r a c t Objectives: Soluble receptor of advanced glycation end-products (sRAGE) may be a predictive biomarker in coronary artery disease (CAD). Patients with acute myocardial infarction (AMI) have higher sRAGE levels compared to healthy subjects. Accordingly, the aim of this study was to investigate the dynamic changes in sRAGE levels during AMI and relationship with cardiac dysfunction. Design and methods: We prospectively included 80 patients with ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI). sRAGE concentrations were measured before pPCI, immediately after pPCI and again on the first and second following days. Left ventricular ejection fraction (LVEF) was evaluated 1–3 days after the pPCI and again at a median of 7 months by echocardiography, and infarct size was measured by cardiac magnetic resonance. Results: sRAGE levels were high in the early phase of AMI; sRAGE levels significantly increased after pPCI compared with sRAGE before pPCI (median ratio: 1.25, 95% CI: 1.15–1.35, P b 0.001), and the increase was observed prior to Troponin I (TnI). sRAGE levels decreased notably the first day after pPCI (median ratio: 0.34, 95% CI: 0.30–0.39, P b 0.001). Peak sRAGE independently associated with long-term cardiac dysfunction estimated by LVEF (P = 0.008). Furthermore, sRAGE measured after pPCI associated with infarct size (P = 0.038). Conclusions: sRAGE levels were high in the early phase rather than in the days after AMI and pPCI. The increase in sRAGE was seen before detectable changes in TnI. In addition, sRAGE was independently associated with long-term cardiac dysfunction. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction The activation and involvement of the receptor of advanced glycation end-products (RAGE) in atherosclerosis and ischemia and reperfusion injury (I/R-injury) have been implied in several studies [1–3]. Soluble RAGE (sRAGE) is an isoform of RAGE, which lacks the intracellular domain and thus intracellular signaling [4]. Instead, sRAGE circulates in the blood and is believed to reflect RAGE and inflammatory activity [5]. In circulation, sRAGE may scavenge RAGE ligands and inhibit RAGE activation [4], though the exact mechanism and main role of sRAGE remain unclear.
⁎ Corresponding author at: The Medical Research Laboratory, Department of Clinical Medicine, Aarhus University and Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Noerrebrogade 44, DK-8000 Aarhus C, Denmark. Fax: +45 78462150. E-mail address: [email protected] (L.J.N. Jensen).
Elevated sRAGE levels are observed in patients with acute myocardial infarction (AMI) [6,7], but patients with chronic coronary artery disease (CAD) display low plasma levels of sRAGE [8,9]. In addition, sRAGE levels positively associated with future CAD [10–12]. Current data on sRAGE and AMI are sparse. The aim of the present study was to evaluate the dynamic changes in sRAGE concentrations during AMI and the association to cardiac dysfunction and infarct size. Methods Study population We prospectively included 80 patients with ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI) at Gentofte University Hospital, Denmark from February 2008 through March 2011 [13]. Inclusion criteria were: significant STsegment elevation in at least two contiguous leads of the electrocardiogram; significant increases in Troponine I (TnI) N 0.03 μg/L; less than
http://dx.doi.org/10.1016/j.clinbiochem.2014.12.022 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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12 hrs from onset of symptoms to pPCI; single vessel disease with a 100% thrombotic occlusion of the left anterior descending branch (LAD) of the left coronary artery; and a successful pPCI resulting in thrombolysis in myocardial infarction (TIMI) 3 flow. Exclusion criteria were: a history of myocardial infarction and/or heart failure. The study was approved by the local scientific ethical committee, and the terms of the Declaration of Helsinki were obtained. Written informed consent was obtained from all participants. Patient characteristics Patients were categorized as diabetics if they were treated with antidiabetic medication at admission. Patients were defined as hypertensive and/or dyslipidemic if they received blood pressure and/or lipid lowering drugs at admission. Estimated glomerular filtration rate (eGFR) was calculated with the simplified MDRD-formula [14]. Treatment with glycoprotein IIb/IIIa inhibitors was used at discretion of the pPCI-operator during pPCI. Echocardiography and cardiac magnetic resonance (CMR)
Plasma concentrations of sRAGE were quantified using an in-house validated time-resolved immunofluorometric assay (TRIFMA) using antibodies and standards from the human RAGE DuoSet (DY1145, R&D Systems, Minneapolis USA). In details, wells were coated with 2 mg/L of mouse anti-human RAGE antibody diluted in phosphate buffered saline (PBS) over night at 4 °C and blocked with 1% Tween20 in PBS for 1 h at room temperature (RT). Samples were diluted 1:10 in a Reagent Diluent containing 0.1% bovine serum albumine (BSA) in PBS and incubated over night at 4 °C. The bound sRAGE was detected with 100 μg/L biotinylated goat anti-human RAGE in Reagent Diluent for 2 h at RT followed by incubation with 10 ng Eu3+-labeled streptavidin (Perkin Elmer, Waltham, Massachusetts, USA) in 100 μl PBS, 0.05% Tween20 containing 25 μM EDTA for 1 h at RT. After each step, wells were washed with 0.05% Tween20 in PBS. Bound europium was detected by adding 200 μl of enhancement solution (Perkin Elmer) and by reading the timeresolved fluorescence on a DELFIA fluorometer (Victor3, Perkin Elmer). The limit of detection was 10 ng/L, and the analytical range was 31– 2000 ng/L. The intra- and interassay coefficients of variation (CV) were below 4% and 12%, respectively. Mean recovery was 87% (range 77– 96%). CV for repeated freeze-thaw cycles of plasma was below 4%.
Conventional two-dimensional echocardiography (using Vivid 7, GE Healthcare, Horten, Norway) was performed 1–3 days after the pPCI and again at a median of 7 months (interquartile range (IQR): 7–8 months) after the infarction. Left ventricular ejection fraction (LVEF) was obtained off-line (using EchoPac, GE Healthcare, Horten, Norway) using the modified Simpson biplane method [15]. Infarct size was assessed with CMR at 1–3 days after pPCI in a subgroup of the patients (43% (n = 34)) as half of the patients were transferred to local hospitals for further treatment, while a few were excluded due to renal failure (eGFR b 60 mL/min/1.73 m2), obesity, metallic implants contra-indicating CMR or claustrophobia. Examinations were performed on a clinical 3.0 T MR scanner (GE). All images were acquired during breath hold and using ECG triggering. Delayed enhancement was assessed on short axis images (gradient echo pulse sequence) in end-diastole covering the whole ventricle (slice thickness 6.0; spacing 4.0) approximately 15 min after injection of bolus contrast (Gadovist 0.2 mmol/kg body weight). To obtain maximum contrast between healthy and necrotic myocardium, the optimal inversionrecovery time (approximately 300 ms) was found. Infarct size was calculated as (volume infarct/volume LV mass) × 100. Images were analyzed off-line using ReportCARDTM 4.0 (GE).
Statistics
Blood samples
Baseline variables are presented in Table 1. No associations between sRAGE and baseline variables were found (data not shown). Infarct size associated negatively with LVEF assessed after a median of 7 months, ß = −1.9956, P = 0.002.
Consecutive blood samples were drawn four times during the study: before the pPCI procedure from the femoral sheath, before injection of contrast fluid (Iomeron®); immediately after pPCI also from the femoral sheath and on the first and second following days from the cubital vein. Blood samples were immediately allocated into containers for coagulation or EDTA containers, following centrifuged at 3,500 g for ten minutes. Serum and plasma were stored in NuncCryo tubes (Nunc, Roskilde, Denmark) at −80 °C until analysis. TnI concentrations were measured with the ARCHITECT STAT Troponin-I (2 K41, Abbott Laboratories, Abbott Park, Illinois, USA), which is a s a two- step chemiluminescent microparticle immunoassay with a limit of detection at 0.01 μg/L, analytical range between 10 and 50.4 μg/L, intra-and interassay CVs below 10%, and the 99th percentile at 0.028 μg/L. C-reactive protein (CRP) levels were estimated by the use of the MULTIGENT CRP Vario (6 K26-30, Abbott Laboratories, Abbott Park, Illinois, USA), an immunoturbidimetric assay with the use of latex particles, which has an analytical range between 0.2 to 320 mg/L and the intra-and interassay CVs below 6%. Creatinine levels were evaluated by an enzymatic assay; the MULTIGENT Creatinine assay (8 L24–31, Abbott Laboratories, Abbott Park, Illinois, USA), which has a measuring range between 8.8 til 3,536.0 μmol/L and intra-and interassay CVs below 3.6%.
Baseline characteristics were described according to Gaussian or non-Gaussian distribution, though dichotomous variables were indicated as percent of total. Symptom-to-balloon time, sRAGE, TnI, and CRP were non-Gaussian distributed and therefore logarithmically transformed using natural logarithm before analysis. Changes in biomarkers were evaluated by Student's paired t-test. Associations with baseline variables, and cardiac dysfunction and infarct size were evaluated in linear regression models. Regression models were validated with plots of residuals, fitted values and leverage. In the multivariate regression analysis, TnI was categorized into quartiles to fit the model. The numbers of missing values were high with regard to CMR and the 4th blood sample, therefore the groups with and without these values were compared according to baseline variables; importantly missing values were due to random effects, and only BMI was significantly lower in patients missing measurements of infarct size. P-values below 5% were considered significant. Stata statistical Software, version 11.2 for Mac (StataCorp LP, College Station, Texas, USA) was used for statistical analysis. Results
Table 1 Baseline characteristics of patients with STEMI. Patients (n = 80) Age (years)a Male sex Diabetes mellitus Hypertension Hypercholesterolemia Current smoking BMI (kg/m2) Creatinine (μmol/L)a eGFR (mL/min)a Use of glycoprotein inhibitor Symptom-to-balloon time (min)b
59 ± 12 85% 6% 19% 11% 45% 26.6 ± 4.2 74 ± 20 97 ± 6 34% 225 (161–269)
Dichotomous variables are presented in %. BMI, body mass index; eGFR, estimated glomerular filtration rate; STEMI, ST-segment elevation myocardial infarction. a Gaussian distributed as mean ± standard deviation. b Non-Gaussian distributed as median (interquartile range).
L.J.N. Jensen et al. / Clinical Biochemistry 48 (2015) 297–301
sRAGE levels significantly increased during AMI from 3,804 (2,522–5,281) (median (IQR)) ng/L before pPCI to 4,535 (3,155– 6,129) (median (IQR)) ng/L immediately after pPCI (median ratio of 1.25 (95% CI: 1.15–1.35), P b 0.001). At the first day after pPCI, sRAGE levels decreased to 1,446 (1,015–2,741) (median (IQR)) ng/L (median ratio of 0.34 (95% CI: 0.30–0.39), P b 0.001), and decreased even further the following day to 1,371 (902–3,121) (median (IQR)) ng/L (median ratio of 0.80 (95% CI: 0.72–0.89), P b 0.001) Fig. 1. Interestingly, sRAGE levels peaked prior to both TnI and CRP Fig. 1a. Peak-sRAGE associated positively with peak-TnI (P = 0.012) whereas, peak-sRAGE and peak-CRP did not (P = 0.129).
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However, CRP associated positively with sRAGE on the first (P = 0.023) and second (P = 0.005) days after pPCI. Increasing sRAGE levels measured immediately after pPCI and on the first day after pPCI, associated with cardiac dysfunction estimated by LVEF in the days after pPCI (P = 0.004 and P = 0.001 respectively), and a median of 7 months after pPCI (P = 0.004) Table 2. Peak sRAGE remained an independent predictor of decreased LVEF at the time of infarction (P = 0.014) and after a median of 7 months (P = 0.008), Table 3. A tendency towards increased infarct size with elevated sRAGE levels was observed and statistical significance at the first day after pPCI (P = 0.038), Table 2.
Fig. 1. a. The biomarkers' soluble receptor of advanced glycation end-products (sRAGE), Troponin I (TnI), and C-reactive protein (CRP) illustrated in a line plot with median values taken before primary percutaneous coronary intervention (pPCI), after pPCI, the day after pPCI and two days after pPCI.● = sRAGE/TnI/CRP before PCI versus after PCI, P b 0.001. ■ = sRAGE/TnI/ CRP after PCI versus the day after PCI, P b 0.001. ▲ = sRAGE/TnI/CRP the day after PCI versus two days after PCI, P b 0.001. b. The biomarkers' soluble receptor of advanced glycation endproducts (sRAGE) (left), Troponin I (TnI) (middle), and C-reactive protein (CRP) (right) illustrated in box plots with median, interquartile range (boxes), 1.5 interquartile range (whiskers) and outliers (dots) before primary percutaneous coronary intervention (pPCI) (1), after pPCI (2), the day after pPCI (3) and two days after pPCI (4).
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Table 2 Univariate regression analysis of repeated measurements of sRAGE and peak sRAGE during AMI and pPCI in relation to infarct size and LVEF in the days and at a median of 7 months after pPCI. Relative infarct size
Log(sRAGE before PCI) Log(sRAGE after PCI) Log(sRAGE one day after PCI) Log(sRAGE two days after PCI) Log(peak sRAGE)
LVEF after pPCI
LVEF at 7 months (median) after pPCI
n
β
P-value
n
β
P-value
n
β
P-value
34 34 34 19 34
1.2 (−1.4–3.9) 1.9 (−0.8–4.5) 3.1 (0.2–6.1) 0.6 (−5.2–6.4) 2.0 (−0.7–4.7)
0.34 0.17 0.038 0.83 0.14
78 79 69 40 79
−2.2 (−5–0.7) −4.6 (−7.7–(−1.5)) −4.5 (−7.2–(−1.9)) −2.3 (−6.7–2.1) −4.5 (−7.6–(−1.4))
0.14 0.004 0.001 0.30 0.005
74 75 65 39 75
−2.4 (−5.5–0.7) −5.1 (−8.6–(−1.7)) −4.3 (−7.2–(−1.4)) −2.2 (−7.1–2.6) −5.3 (−8.7–(−1.9))
0.12 0.004 0.004 0.36 0.003
Data are presented as coefficient of regression equation (ß) with 95% confidence intervals (CI). AMI, acute myocardial infarction; LVEF, left ventricular ejection fraction; pPCI, primary percutaneous intervention (pPCI); sRAGE, soluble receptor of advanced glycation end-products.
Discussion In this study, we show that sRAGE levels during AMI follow a course similar to TnI, but interestingly sRAGE levels peaked prior to both TnI and CRP. Peak sRAGE concentration associated independently with cardiac dysfunction as assessed by echocardiographic LVEF both at the time of AMI and after a median of 7 months. Several different ligands bind to and activate RAGE [16,17], and mediate inflammatory and oxidative activity contributing to the development of vascular pathogenesis [18,19]. sRAGE, a 46 KD protein, exists in either a truncated or a cleaved form of the full-length RAGE protein [5,20,21]. sRAGE lacks the intracellular part of the protein thus no intracellular signaling appears after binding of RAGE ligands [4]. Thus sRAGE is suggested to acts as a decoy protein of RAGE ligands and possibly reflects RAGE and inflammatory activity [5,22,23]. Few clinical studies have investigated sRAGE levels in relation to AMI. Two studies found significantly increased sRAGE levels in patients with acute coronary syndrome compared to healthy controls [6,7], whereas a third study found the opposite [24]. The altered levels of sRAGE in our study may explain the opposing results; the different times of blood sample collection makes it difficult to compare the studies. Circulating sRAGE is lower in patients with stabile CAD when compared to controls [8,9]. Two prospective studies found that lower sRAGE levels associated independently with future cardiovascular events [12, 25]. First demonstrated in a smaller cohort of non-diabetic patients suspected of CAD with 48 months of follow-up [25] and recently, in 1,201 healthy participants from the Atherosclerosis Risk in Communities Study; lower sRAGE levels independently associated with risk of CAD and all-cause mortality during 18 years of follow-up [12]. Possibly, the low sRAGE level is due to the decoying effect of RAGE ligands in the stabile phase of CAD. On the contrary, a rapid increase in sRAGE is triggered during AMI, reflecting an inflammatory response. In the present
Table 3 Multivariate regression analysis of peak levels of sRAGE in relation to LVEF in the days and at a median of 7 months after pPCI. LVEF after pPCI Log(peak sRAGE) β (95% CI) Model 1: Model 2: Model 3: Model 4: Model 5: Model 6:
−4.5 (−7.6–(−1.4)) −4.3 (−7.5(−1.1)) −4.3 (−7.6–(−1.1)) −4.3 (−7.6–(−0.9)) −3.7 (−7.0–(−0.4)) −4.0 (−7.4–(−0.6))
LVEF at 7 months (median) after pPCI P-value β (95% CI) 0.005 0.009 0.01 0.014 0.03 0.014
−5.3 (−8.7–(−1.9)) −5.4 (−8.9–(−2.0)) −5.8 (−9.3–(−2.3)) −5.7 (−9.3–(−2.1)) −4.8 (−8.4–(−1.3)) −5.0 (−8.6–(1.3))
P-value 0.003 0.003 0.001 0.002 0.008 0.008
Data are presented as coefficient of regression equation (ß) with 95% confidence intervals (CI). Model 1: univariate; model 2: model 1 + age; model 3: model 2 + gender; model 4: model 3 + eGFR; model 5: model 4 + TnI (after pPCI); model 6: model 5 + CRP (after PCI). CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; LVEF, left ventricular ejection fraction; pPCI, primary percutaneous coronary intervention; sRAGE, soluble receptor of advanced glycation end-products; TnI, Troponin I. Stepwise adjustment with age, gender, eGFR, TnI and CRP.
study, the sRAGE levels were high in the early phase rather than in the days of the infarction. sRAGE levels increased after pPCI, which may suggest an additive increased RAGE activity in relation to reperfusion as a part of I/R-injury, possibly released from vascular and inflammatory cells in the damaged myocardium. The troponins and to a lesser extent the creatinine kinase (CK) are the most specific biomarkers in relation to AMI, all though, alterations in the biomarkers are mostly reliable for diagnosis [26]. The high levels of sRAGE in the early phase of AMI may suggest sRAGE as an additional biomarker of AMI. Importantly, the notable decrease seen on the day after pPCI also suggests that sRAGE may be valuable in the assessment of I/R-injury and re-infarction. sRAGE levels change rapidly during AMI, which is why time point of sRAGE evaluation is extremely important in future studies of sRAGE. Some studies imply that RAGE and sRAGE have high affinity for heparin binding [27,28], therefore it cannot be ruled out that heparin administration may have influence on sRAGE levels. LVEF may indirectly indicate the extent of myocardial damage [29]. In the present study, relative infarct size associated with reduced long-term LVEF measured three to six months after AMI. This association was not seen when LVEF was measured in the first days after AMI, however this may be due to myocardial stunning, as heart function stabilizes during recovery [30]. Increasing sRAGE levels after pPCI and the day after pPCI associated significantly with lower LVEF, and peak sRAGE negatively associated with LVEF when adjusted for several confounders. This is in accordance with the findings of Koyama et al. who demonstrated that increasing sRAGE levels associated with decreasing LVEF in patients with heart failure. Additionally, sRAGE also associated with increased hospitalization and all-cause mortality [31]. The association with cardiac dysfunction may propose sRAGE as a helpful and easy method of early prediction of cardiac dysfunction after AMI. It is well known that CRP is associated with CAD [32,33]. We found a positive association between sRAGE and CRP levels measured one and two days after pPCI, indicating a relationship with systemic inflammation, however this needs further investigation. Limitations: The present study only included eighty patients; accordingly results must be interpreted with caution. Additionally, only 43% (n = 34) of the patients underwent a complete CMR-scan, possibly weakening the association between sRAGE and infarct size. Control samples drawn before AMI were not available but would have been valuable. The contribution of the pPCI procedure to the sRAGE levels was not possible to differentiate. The assay used for measurement of sRAGE is known to measure total sRAGE levels, however, it was not possible to differentiate between secreted sRAGE and cleaved RAGE, although this may be of relevance.
Conclusion High levels of sRAGE were found in the early phase of AMI with a rapid decrease already one day after pPCI. The elevated level of sRAGE was seen prior to a similar increase in TnI level and may suggest sRAGE as an additional biomarker of AMI. In addition, sRAGE
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independently associated with long-term cardiac dysfunction, but the predictive value needs further investigation. Research funding This work was supported by grants from the Helga and Peter Korning Foundation, Viby, Denmark; the Eva and Henry Frænkels Memorial Foundation, Holte, Denmark; The Danish Council for Independent Research; The Danish Diabetes Association Odense, Denmark; and the Lundbeck Foundation, Hellerup, Denmark. Author's conflict of interest disclosure None declared. Acknowledgements Kirsten Nyborg Rasmussen is thanked for her excellent technical assistance. References [1] Aleshin A, Ananthakrishnan R, Li Q, Rosario R, Lu Y, Qu W, et al. Rage modulates myocardial injury consequent to lad infarction via impact on jnk and stat signaling in a murine model. Am J Physiol Heart Circ Physiol 2008;294:H1823–32. [2] Harja E, Bu DX, Hudson BI, Chang JS, Shen X, Hallam K, et al. Vascular and inflammatory stresses mediate atherosclerosis via rage and its ligands in apoe-/- mice. J Clin Invest 2008;118:183–94. [3] Soro-Paavonen A, Watson AM, Li J, Paavonen K, Koitka A, Calkin AC, et al. Receptor for advanced glycation end products (rage) deficiency attenuates the development of atherosclerosis in diabetes. Diabetes 2008;57:2461–9. [4] Yonekura H, Yamamoto Y, Sakurai S, Petrova RG, Abedin MJ, Li H, et al. Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetesinduced vascular injury. Biochem J 2003;370:1097–109. [5] Raucci A, Cugusi S, Antonelli A, Barabino SM, Monti L, Bierhaus A, et al. A soluble form of the receptor for advanced glycation endproducts (rage) is produced by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (adam10). FASEB J 2008;22:3716–27. [6] Cai XY, Lu L, Wang YN, Jin C, Zhang RY, Zhang Q, et al. Association of increased s100b, s100a6 and s100p in serum levels with acute coronary syndrome and also with the severity of myocardial infarction in cardiac tissue of rat models with ischemiareperfusion injury. Atherosclerosis 2011;217:536–42. [7] Park HJ, Baek JY, Shin WS, Kim DB, Jang SW, Shin DI, et al. Soluble receptor of advanced glycated endproducts is associated with plaque vulnerability in patients with acute myocardial infarction. Circ J 2011;75:1685–90. [8] Falcone C, Emanuele E, D'Angelo A, Buzzi MP, Belvito C, Cuccia M, et al. Plasma levels of soluble receptor for advanced glycation end products and coronary artery disease in nondiabetic men. Arterioscler Thromb Vasc Biol 2005;25:1032–7. [9] Mahajan N, Malik N, Bahl A, Dhawan V. Receptor for advanced glycation end products (rage) and its inflammatory ligand en-rage in non-diabetic subjects with premature coronary artery disease. Atherosclerosis 2009;207:597–602. [10] Colhoun HM, Betteridge DJ, Durrington P, Hitman G, Neil A, Livingstone S, et al. Total soluble and endogenous secretory receptor for advanced glycation end products as predictive biomarkers of coronary heart disease risk in patients with type 2 diabetes: an analysis from the cards trial. Diabetes 2011;60:2379–85. [11] Nin JW, Ferreira I, Schalkwijk CG, Prins MH, Chaturvedi N, Fuller JH, et al. Levels of soluble receptor for age are cross-sectionally associated with cardiovascular disease in type 1 diabetes, and this association is partially mediated by endothelial and renal dysfunction and by low-grade inflammation: the eurodiab prospective complications study. Diabetologia 2009;52:705–14. [12] Selvin E, Halushka M, Rawlings A, Hoogeveen RC, Ballantyne CM, Coresh J, et al. Srage and risk of diabetes, cardiovascular disease and death. Diabetes 2013;62: 2116–21. [13] Lindberg S, Jensen JS, Hoffmann S, Iversen AZ, Pedersen SH, Møgelvang R, et al. Osteoprotegerin levels change during stemi and reflect cardiac function. Can J Cardiol 2014;30:1523–8.
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Dynamic changes in sRAGE levels and relationship with cardiac function in STEMI patients Louise J.N. Jensen a,b,⁎, Søren Lindberg c, Søren Hoffmann c, Allan Z. Iversen c, Sune H. Pedersen c, Rasmus Møgelvang c, Søren Galatius c, Allan Flyvbjerg a,b, Jan S. Jensen c,d, Mette Bjerre a,b a
The Medical Research Laboratory, Department of Clinical Medicine, Aarhus University, Noerrebrogade 44, DK-8000 Aarhus C, Denmark Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Noerrebrogade 44, DK-8000 Aarhus C, Denmark Department of Cardiology P, Gentofte University Hospital, Niels Andersens Vej 65, DK-2900 Hellerup, Denmark d Institute of Clinical Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen N, Denmark b c
a r t i c l e
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Article history: Received 29 August 2014 Received in revised form 19 November 2014 Accepted 23 December 2014 Available online 3 January 2015 Keywords: Soluble receptor of advanced glycation end-products sRAGE Biomarker Acute myocardial infarction Primary percutaneous coronary intervention Left ventricular ejection fraction
a b s t r a c t Objectives: Soluble receptor of advanced glycation end-products (sRAGE) may be a predictive biomarker in coronary artery disease (CAD). Patients with acute myocardial infarction (AMI) have higher sRAGE levels compared to healthy subjects. Accordingly, the aim of this study was to investigate the dynamic changes in sRAGE levels during AMI and relationship with cardiac dysfunction. Design and methods: We prospectively included 80 patients with ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI). sRAGE concentrations were measured before pPCI, immediately after pPCI and again on the first and second following days. Left ventricular ejection fraction (LVEF) was evaluated 1–3 days after the pPCI and again at a median of 7 months by echocardiography, and infarct size was measured by cardiac magnetic resonance. Results: sRAGE levels were high in the early phase of AMI; sRAGE levels significantly increased after pPCI compared with sRAGE before pPCI (median ratio: 1.25, 95% CI: 1.15–1.35, P b 0.001), and the increase was observed prior to Troponin I (TnI). sRAGE levels decreased notably the first day after pPCI (median ratio: 0.34, 95% CI: 0.30–0.39, P b 0.001). Peak sRAGE independently associated with long-term cardiac dysfunction estimated by LVEF (P = 0.008). Furthermore, sRAGE measured after pPCI associated with infarct size (P = 0.038). Conclusions: sRAGE levels were high in the early phase rather than in the days after AMI and pPCI. The increase in sRAGE was seen before detectable changes in TnI. In addition, sRAGE was independently associated with long-term cardiac dysfunction. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction The activation and involvement of the receptor of advanced glycation end-products (RAGE) in atherosclerosis and ischemia and reperfusion injury (I/R-injury) have been implied in several studies [1–3]. Soluble RAGE (sRAGE) is an isoform of RAGE, which lacks the intracellular domain and thus intracellular signaling [4]. Instead, sRAGE circulates in the blood and is believed to reflect RAGE and inflammatory activity [5]. In circulation, sRAGE may scavenge RAGE ligands and inhibit RAGE activation [4], though the exact mechanism and main role of sRAGE remain unclear.
⁎ Corresponding author at: The Medical Research Laboratory, Department of Clinical Medicine, Aarhus University and Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Noerrebrogade 44, DK-8000 Aarhus C, Denmark. Fax: +45 78462150. E-mail address: [email protected] (L.J.N. Jensen).
Elevated sRAGE levels are observed in patients with acute myocardial infarction (AMI) [6,7], but patients with chronic coronary artery disease (CAD) display low plasma levels of sRAGE [8,9]. In addition, sRAGE levels positively associated with future CAD [10–12]. Current data on sRAGE and AMI are sparse. The aim of the present study was to evaluate the dynamic changes in sRAGE concentrations during AMI and the association to cardiac dysfunction and infarct size. Methods Study population We prospectively included 80 patients with ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI) at Gentofte University Hospital, Denmark from February 2008 through March 2011 [13]. Inclusion criteria were: significant STsegment elevation in at least two contiguous leads of the electrocardiogram; significant increases in Troponine I (TnI) N 0.03 μg/L; less than
http://dx.doi.org/10.1016/j.clinbiochem.2014.12.022 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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12 hrs from onset of symptoms to pPCI; single vessel disease with a 100% thrombotic occlusion of the left anterior descending branch (LAD) of the left coronary artery; and a successful pPCI resulting in thrombolysis in myocardial infarction (TIMI) 3 flow. Exclusion criteria were: a history of myocardial infarction and/or heart failure. The study was approved by the local scientific ethical committee, and the terms of the Declaration of Helsinki were obtained. Written informed consent was obtained from all participants. Patient characteristics Patients were categorized as diabetics if they were treated with antidiabetic medication at admission. Patients were defined as hypertensive and/or dyslipidemic if they received blood pressure and/or lipid lowering drugs at admission. Estimated glomerular filtration rate (eGFR) was calculated with the simplified MDRD-formula [14]. Treatment with glycoprotein IIb/IIIa inhibitors was used at discretion of the pPCI-operator during pPCI. Echocardiography and cardiac magnetic resonance (CMR)
Plasma concentrations of sRAGE were quantified using an in-house validated time-resolved immunofluorometric assay (TRIFMA) using antibodies and standards from the human RAGE DuoSet (DY1145, R&D Systems, Minneapolis USA). In details, wells were coated with 2 mg/L of mouse anti-human RAGE antibody diluted in phosphate buffered saline (PBS) over night at 4 °C and blocked with 1% Tween20 in PBS for 1 h at room temperature (RT). Samples were diluted 1:10 in a Reagent Diluent containing 0.1% bovine serum albumine (BSA) in PBS and incubated over night at 4 °C. The bound sRAGE was detected with 100 μg/L biotinylated goat anti-human RAGE in Reagent Diluent for 2 h at RT followed by incubation with 10 ng Eu3+-labeled streptavidin (Perkin Elmer, Waltham, Massachusetts, USA) in 100 μl PBS, 0.05% Tween20 containing 25 μM EDTA for 1 h at RT. After each step, wells were washed with 0.05% Tween20 in PBS. Bound europium was detected by adding 200 μl of enhancement solution (Perkin Elmer) and by reading the timeresolved fluorescence on a DELFIA fluorometer (Victor3, Perkin Elmer). The limit of detection was 10 ng/L, and the analytical range was 31– 2000 ng/L. The intra- and interassay coefficients of variation (CV) were below 4% and 12%, respectively. Mean recovery was 87% (range 77– 96%). CV for repeated freeze-thaw cycles of plasma was below 4%.
Conventional two-dimensional echocardiography (using Vivid 7, GE Healthcare, Horten, Norway) was performed 1–3 days after the pPCI and again at a median of 7 months (interquartile range (IQR): 7–8 months) after the infarction. Left ventricular ejection fraction (LVEF) was obtained off-line (using EchoPac, GE Healthcare, Horten, Norway) using the modified Simpson biplane method [15]. Infarct size was assessed with CMR at 1–3 days after pPCI in a subgroup of the patients (43% (n = 34)) as half of the patients were transferred to local hospitals for further treatment, while a few were excluded due to renal failure (eGFR b 60 mL/min/1.73 m2), obesity, metallic implants contra-indicating CMR or claustrophobia. Examinations were performed on a clinical 3.0 T MR scanner (GE). All images were acquired during breath hold and using ECG triggering. Delayed enhancement was assessed on short axis images (gradient echo pulse sequence) in end-diastole covering the whole ventricle (slice thickness 6.0; spacing 4.0) approximately 15 min after injection of bolus contrast (Gadovist 0.2 mmol/kg body weight). To obtain maximum contrast between healthy and necrotic myocardium, the optimal inversionrecovery time (approximately 300 ms) was found. Infarct size was calculated as (volume infarct/volume LV mass) × 100. Images were analyzed off-line using ReportCARDTM 4.0 (GE).
Statistics
Blood samples
Baseline variables are presented in Table 1. No associations between sRAGE and baseline variables were found (data not shown). Infarct size associated negatively with LVEF assessed after a median of 7 months, ß = −1.9956, P = 0.002.
Consecutive blood samples were drawn four times during the study: before the pPCI procedure from the femoral sheath, before injection of contrast fluid (Iomeron®); immediately after pPCI also from the femoral sheath and on the first and second following days from the cubital vein. Blood samples were immediately allocated into containers for coagulation or EDTA containers, following centrifuged at 3,500 g for ten minutes. Serum and plasma were stored in NuncCryo tubes (Nunc, Roskilde, Denmark) at −80 °C until analysis. TnI concentrations were measured with the ARCHITECT STAT Troponin-I (2 K41, Abbott Laboratories, Abbott Park, Illinois, USA), which is a s a two- step chemiluminescent microparticle immunoassay with a limit of detection at 0.01 μg/L, analytical range between 10 and 50.4 μg/L, intra-and interassay CVs below 10%, and the 99th percentile at 0.028 μg/L. C-reactive protein (CRP) levels were estimated by the use of the MULTIGENT CRP Vario (6 K26-30, Abbott Laboratories, Abbott Park, Illinois, USA), an immunoturbidimetric assay with the use of latex particles, which has an analytical range between 0.2 to 320 mg/L and the intra-and interassay CVs below 6%. Creatinine levels were evaluated by an enzymatic assay; the MULTIGENT Creatinine assay (8 L24–31, Abbott Laboratories, Abbott Park, Illinois, USA), which has a measuring range between 8.8 til 3,536.0 μmol/L and intra-and interassay CVs below 3.6%.
Baseline characteristics were described according to Gaussian or non-Gaussian distribution, though dichotomous variables were indicated as percent of total. Symptom-to-balloon time, sRAGE, TnI, and CRP were non-Gaussian distributed and therefore logarithmically transformed using natural logarithm before analysis. Changes in biomarkers were evaluated by Student's paired t-test. Associations with baseline variables, and cardiac dysfunction and infarct size were evaluated in linear regression models. Regression models were validated with plots of residuals, fitted values and leverage. In the multivariate regression analysis, TnI was categorized into quartiles to fit the model. The numbers of missing values were high with regard to CMR and the 4th blood sample, therefore the groups with and without these values were compared according to baseline variables; importantly missing values were due to random effects, and only BMI was significantly lower in patients missing measurements of infarct size. P-values below 5% were considered significant. Stata statistical Software, version 11.2 for Mac (StataCorp LP, College Station, Texas, USA) was used for statistical analysis. Results
Table 1 Baseline characteristics of patients with STEMI. Patients (n = 80) Age (years)a Male sex Diabetes mellitus Hypertension Hypercholesterolemia Current smoking BMI (kg/m2) Creatinine (μmol/L)a eGFR (mL/min)a Use of glycoprotein inhibitor Symptom-to-balloon time (min)b
59 ± 12 85% 6% 19% 11% 45% 26.6 ± 4.2 74 ± 20 97 ± 6 34% 225 (161–269)
Dichotomous variables are presented in %. BMI, body mass index; eGFR, estimated glomerular filtration rate; STEMI, ST-segment elevation myocardial infarction. a Gaussian distributed as mean ± standard deviation. b Non-Gaussian distributed as median (interquartile range).
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sRAGE levels significantly increased during AMI from 3,804 (2,522–5,281) (median (IQR)) ng/L before pPCI to 4,535 (3,155– 6,129) (median (IQR)) ng/L immediately after pPCI (median ratio of 1.25 (95% CI: 1.15–1.35), P b 0.001). At the first day after pPCI, sRAGE levels decreased to 1,446 (1,015–2,741) (median (IQR)) ng/L (median ratio of 0.34 (95% CI: 0.30–0.39), P b 0.001), and decreased even further the following day to 1,371 (902–3,121) (median (IQR)) ng/L (median ratio of 0.80 (95% CI: 0.72–0.89), P b 0.001) Fig. 1. Interestingly, sRAGE levels peaked prior to both TnI and CRP Fig. 1a. Peak-sRAGE associated positively with peak-TnI (P = 0.012) whereas, peak-sRAGE and peak-CRP did not (P = 0.129).
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However, CRP associated positively with sRAGE on the first (P = 0.023) and second (P = 0.005) days after pPCI. Increasing sRAGE levels measured immediately after pPCI and on the first day after pPCI, associated with cardiac dysfunction estimated by LVEF in the days after pPCI (P = 0.004 and P = 0.001 respectively), and a median of 7 months after pPCI (P = 0.004) Table 2. Peak sRAGE remained an independent predictor of decreased LVEF at the time of infarction (P = 0.014) and after a median of 7 months (P = 0.008), Table 3. A tendency towards increased infarct size with elevated sRAGE levels was observed and statistical significance at the first day after pPCI (P = 0.038), Table 2.
Fig. 1. a. The biomarkers' soluble receptor of advanced glycation end-products (sRAGE), Troponin I (TnI), and C-reactive protein (CRP) illustrated in a line plot with median values taken before primary percutaneous coronary intervention (pPCI), after pPCI, the day after pPCI and two days after pPCI.● = sRAGE/TnI/CRP before PCI versus after PCI, P b 0.001. ■ = sRAGE/TnI/ CRP after PCI versus the day after PCI, P b 0.001. ▲ = sRAGE/TnI/CRP the day after PCI versus two days after PCI, P b 0.001. b. The biomarkers' soluble receptor of advanced glycation endproducts (sRAGE) (left), Troponin I (TnI) (middle), and C-reactive protein (CRP) (right) illustrated in box plots with median, interquartile range (boxes), 1.5 interquartile range (whiskers) and outliers (dots) before primary percutaneous coronary intervention (pPCI) (1), after pPCI (2), the day after pPCI (3) and two days after pPCI (4).
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Table 2 Univariate regression analysis of repeated measurements of sRAGE and peak sRAGE during AMI and pPCI in relation to infarct size and LVEF in the days and at a median of 7 months after pPCI. Relative infarct size
Log(sRAGE before PCI) Log(sRAGE after PCI) Log(sRAGE one day after PCI) Log(sRAGE two days after PCI) Log(peak sRAGE)
LVEF after pPCI
LVEF at 7 months (median) after pPCI
n
β
P-value
n
β
P-value
n
β
P-value
34 34 34 19 34
1.2 (−1.4–3.9) 1.9 (−0.8–4.5) 3.1 (0.2–6.1) 0.6 (−5.2–6.4) 2.0 (−0.7–4.7)
0.34 0.17 0.038 0.83 0.14
78 79 69 40 79
−2.2 (−5–0.7) −4.6 (−7.7–(−1.5)) −4.5 (−7.2–(−1.9)) −2.3 (−6.7–2.1) −4.5 (−7.6–(−1.4))
0.14 0.004 0.001 0.30 0.005
74 75 65 39 75
−2.4 (−5.5–0.7) −5.1 (−8.6–(−1.7)) −4.3 (−7.2–(−1.4)) −2.2 (−7.1–2.6) −5.3 (−8.7–(−1.9))
0.12 0.004 0.004 0.36 0.003
Data are presented as coefficient of regression equation (ß) with 95% confidence intervals (CI). AMI, acute myocardial infarction; LVEF, left ventricular ejection fraction; pPCI, primary percutaneous intervention (pPCI); sRAGE, soluble receptor of advanced glycation end-products.
Discussion In this study, we show that sRAGE levels during AMI follow a course similar to TnI, but interestingly sRAGE levels peaked prior to both TnI and CRP. Peak sRAGE concentration associated independently with cardiac dysfunction as assessed by echocardiographic LVEF both at the time of AMI and after a median of 7 months. Several different ligands bind to and activate RAGE [16,17], and mediate inflammatory and oxidative activity contributing to the development of vascular pathogenesis [18,19]. sRAGE, a 46 KD protein, exists in either a truncated or a cleaved form of the full-length RAGE protein [5,20,21]. sRAGE lacks the intracellular part of the protein thus no intracellular signaling appears after binding of RAGE ligands [4]. Thus sRAGE is suggested to acts as a decoy protein of RAGE ligands and possibly reflects RAGE and inflammatory activity [5,22,23]. Few clinical studies have investigated sRAGE levels in relation to AMI. Two studies found significantly increased sRAGE levels in patients with acute coronary syndrome compared to healthy controls [6,7], whereas a third study found the opposite [24]. The altered levels of sRAGE in our study may explain the opposing results; the different times of blood sample collection makes it difficult to compare the studies. Circulating sRAGE is lower in patients with stabile CAD when compared to controls [8,9]. Two prospective studies found that lower sRAGE levels associated independently with future cardiovascular events [12, 25]. First demonstrated in a smaller cohort of non-diabetic patients suspected of CAD with 48 months of follow-up [25] and recently, in 1,201 healthy participants from the Atherosclerosis Risk in Communities Study; lower sRAGE levels independently associated with risk of CAD and all-cause mortality during 18 years of follow-up [12]. Possibly, the low sRAGE level is due to the decoying effect of RAGE ligands in the stabile phase of CAD. On the contrary, a rapid increase in sRAGE is triggered during AMI, reflecting an inflammatory response. In the present
Table 3 Multivariate regression analysis of peak levels of sRAGE in relation to LVEF in the days and at a median of 7 months after pPCI. LVEF after pPCI Log(peak sRAGE) β (95% CI) Model 1: Model 2: Model 3: Model 4: Model 5: Model 6:
−4.5 (−7.6–(−1.4)) −4.3 (−7.5(−1.1)) −4.3 (−7.6–(−1.1)) −4.3 (−7.6–(−0.9)) −3.7 (−7.0–(−0.4)) −4.0 (−7.4–(−0.6))
LVEF at 7 months (median) after pPCI P-value β (95% CI) 0.005 0.009 0.01 0.014 0.03 0.014
−5.3 (−8.7–(−1.9)) −5.4 (−8.9–(−2.0)) −5.8 (−9.3–(−2.3)) −5.7 (−9.3–(−2.1)) −4.8 (−8.4–(−1.3)) −5.0 (−8.6–(1.3))
P-value 0.003 0.003 0.001 0.002 0.008 0.008
Data are presented as coefficient of regression equation (ß) with 95% confidence intervals (CI). Model 1: univariate; model 2: model 1 + age; model 3: model 2 + gender; model 4: model 3 + eGFR; model 5: model 4 + TnI (after pPCI); model 6: model 5 + CRP (after PCI). CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; LVEF, left ventricular ejection fraction; pPCI, primary percutaneous coronary intervention; sRAGE, soluble receptor of advanced glycation end-products; TnI, Troponin I. Stepwise adjustment with age, gender, eGFR, TnI and CRP.
study, the sRAGE levels were high in the early phase rather than in the days of the infarction. sRAGE levels increased after pPCI, which may suggest an additive increased RAGE activity in relation to reperfusion as a part of I/R-injury, possibly released from vascular and inflammatory cells in the damaged myocardium. The troponins and to a lesser extent the creatinine kinase (CK) are the most specific biomarkers in relation to AMI, all though, alterations in the biomarkers are mostly reliable for diagnosis [26]. The high levels of sRAGE in the early phase of AMI may suggest sRAGE as an additional biomarker of AMI. Importantly, the notable decrease seen on the day after pPCI also suggests that sRAGE may be valuable in the assessment of I/R-injury and re-infarction. sRAGE levels change rapidly during AMI, which is why time point of sRAGE evaluation is extremely important in future studies of sRAGE. Some studies imply that RAGE and sRAGE have high affinity for heparin binding [27,28], therefore it cannot be ruled out that heparin administration may have influence on sRAGE levels. LVEF may indirectly indicate the extent of myocardial damage [29]. In the present study, relative infarct size associated with reduced long-term LVEF measured three to six months after AMI. This association was not seen when LVEF was measured in the first days after AMI, however this may be due to myocardial stunning, as heart function stabilizes during recovery [30]. Increasing sRAGE levels after pPCI and the day after pPCI associated significantly with lower LVEF, and peak sRAGE negatively associated with LVEF when adjusted for several confounders. This is in accordance with the findings of Koyama et al. who demonstrated that increasing sRAGE levels associated with decreasing LVEF in patients with heart failure. Additionally, sRAGE also associated with increased hospitalization and all-cause mortality [31]. The association with cardiac dysfunction may propose sRAGE as a helpful and easy method of early prediction of cardiac dysfunction after AMI. It is well known that CRP is associated with CAD [32,33]. We found a positive association between sRAGE and CRP levels measured one and two days after pPCI, indicating a relationship with systemic inflammation, however this needs further investigation. Limitations: The present study only included eighty patients; accordingly results must be interpreted with caution. Additionally, only 43% (n = 34) of the patients underwent a complete CMR-scan, possibly weakening the association between sRAGE and infarct size. Control samples drawn before AMI were not available but would have been valuable. The contribution of the pPCI procedure to the sRAGE levels was not possible to differentiate. The assay used for measurement of sRAGE is known to measure total sRAGE levels, however, it was not possible to differentiate between secreted sRAGE and cleaved RAGE, although this may be of relevance.
Conclusion High levels of sRAGE were found in the early phase of AMI with a rapid decrease already one day after pPCI. The elevated level of sRAGE was seen prior to a similar increase in TnI level and may suggest sRAGE as an additional biomarker of AMI. In addition, sRAGE
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independently associated with long-term cardiac dysfunction, but the predictive value needs further investigation. Research funding This work was supported by grants from the Helga and Peter Korning Foundation, Viby, Denmark; the Eva and Henry Frænkels Memorial Foundation, Holte, Denmark; The Danish Council for Independent Research; The Danish Diabetes Association Odense, Denmark; and the Lundbeck Foundation, Hellerup, Denmark. Author's conflict of interest disclosure None declared. Acknowledgements Kirsten Nyborg Rasmussen is thanked for her excellent technical assistance. References [1] Aleshin A, Ananthakrishnan R, Li Q, Rosario R, Lu Y, Qu W, et al. Rage modulates myocardial injury consequent to lad infarction via impact on jnk and stat signaling in a murine model. Am J Physiol Heart Circ Physiol 2008;294:H1823–32. [2] Harja E, Bu DX, Hudson BI, Chang JS, Shen X, Hallam K, et al. Vascular and inflammatory stresses mediate atherosclerosis via rage and its ligands in apoe-/- mice. J Clin Invest 2008;118:183–94. [3] Soro-Paavonen A, Watson AM, Li J, Paavonen K, Koitka A, Calkin AC, et al. Receptor for advanced glycation end products (rage) deficiency attenuates the development of atherosclerosis in diabetes. Diabetes 2008;57:2461–9. [4] Yonekura H, Yamamoto Y, Sakurai S, Petrova RG, Abedin MJ, Li H, et al. Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetesinduced vascular injury. Biochem J 2003;370:1097–109. [5] Raucci A, Cugusi S, Antonelli A, Barabino SM, Monti L, Bierhaus A, et al. A soluble form of the receptor for advanced glycation endproducts (rage) is produced by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (adam10). FASEB J 2008;22:3716–27. [6] Cai XY, Lu L, Wang YN, Jin C, Zhang RY, Zhang Q, et al. Association of increased s100b, s100a6 and s100p in serum levels with acute coronary syndrome and also with the severity of myocardial infarction in cardiac tissue of rat models with ischemiareperfusion injury. Atherosclerosis 2011;217:536–42. [7] Park HJ, Baek JY, Shin WS, Kim DB, Jang SW, Shin DI, et al. Soluble receptor of advanced glycated endproducts is associated with plaque vulnerability in patients with acute myocardial infarction. Circ J 2011;75:1685–90. [8] Falcone C, Emanuele E, D'Angelo A, Buzzi MP, Belvito C, Cuccia M, et al. Plasma levels of soluble receptor for advanced glycation end products and coronary artery disease in nondiabetic men. Arterioscler Thromb Vasc Biol 2005;25:1032–7. [9] Mahajan N, Malik N, Bahl A, Dhawan V. Receptor for advanced glycation end products (rage) and its inflammatory ligand en-rage in non-diabetic subjects with premature coronary artery disease. Atherosclerosis 2009;207:597–602. [10] Colhoun HM, Betteridge DJ, Durrington P, Hitman G, Neil A, Livingstone S, et al. Total soluble and endogenous secretory receptor for advanced glycation end products as predictive biomarkers of coronary heart disease risk in patients with type 2 diabetes: an analysis from the cards trial. Diabetes 2011;60:2379–85. [11] Nin JW, Ferreira I, Schalkwijk CG, Prins MH, Chaturvedi N, Fuller JH, et al. Levels of soluble receptor for age are cross-sectionally associated with cardiovascular disease in type 1 diabetes, and this association is partially mediated by endothelial and renal dysfunction and by low-grade inflammation: the eurodiab prospective complications study. Diabetologia 2009;52:705–14. [12] Selvin E, Halushka M, Rawlings A, Hoogeveen RC, Ballantyne CM, Coresh J, et al. Srage and risk of diabetes, cardiovascular disease and death. Diabetes 2013;62: 2116–21. [13] Lindberg S, Jensen JS, Hoffmann S, Iversen AZ, Pedersen SH, Møgelvang R, et al. Osteoprotegerin levels change during stemi and reflect cardiac function. Can J Cardiol 2014;30:1523–8.
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