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Circulating Soluble Receptor for Advanced Glycation End Products is Inversely Correlated to Oxidized Low-density Lipoproteins in Asymptomatic Subjects K Kotani, R Caccavello, N Taniguchi and A Gugliucci Journal of International Medical Research 2012 40: 1878 DOI: 10.1177/030006051204000527 The online version of this article can be found at: http://imr.sagepub.com/content/40/5/1878

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The Journal of International Medical Research 2012; 40: 1878 – 1883 [first published online as 40(4) 2]

Circulating Soluble Receptor for Advanced Glycation End Products is Inversely Correlated to Oxidized Low-density Lipoproteins in Asymptomatic Subjects K KOTANI1,2, R CACCAVELLO2, N TANIGUCHI1

AND

A GUGLIUCCI2

1

Department of Clinical Laboratory Medicine, Jichi Medical University, Tochigi, Japan; 2 Glycation, Oxidation and Disease Laboratory, Touro University–California, Vallejo, California, USA

OBJECTIVE: There is growing evidence that circulating soluble receptor for advanced glycation end products (sRAGE) exerts antiatherogenic effects as a decoy receptor that abolishes RAGE signalling. A previous study reported that oxidized low-density lipoprotein (oxLDL) can be one of the RAGE ligands. The present cross-sectional study investigated the clinical association between sRAGE and oxLDL in humans. METHODS: Serum levels of the conventional atherosclerotic risk factors, sRAGE and malondialdehyde-modified low-density lipoprotein (MDA–LDL) were analysed in asymptomatic subjects; MDA– LDL was measured as a biomarker of

oxLDL. RESULTS: Mean serum levels of sRAGE and MDA–LDL were 1101 ng/l and 57.6 IU/l, respectively, in 33 subjects of mean age 65 years. Simple linear regression analysis showed a significant inverse correlation between sRAGE and MDA–LDL. Stepwise multiple linear regression analysis confirmed MDA–LDL to be independently, significantly and inversely correlated with sRAGE. CONCLUSIONS: An independent, significant and inverse correlation was shown to exist between circulating levels of sRAGE and oxLDL (MDA–LDL), which suggests that part of the antiatherosclerotic effects of sRAGE may be related to oxLDL quenching.

KEY WORDS: OXIDATIVE STRESS; OXIDIZED LIPOPROTEIN; ADVANCED GLYCATION END PRODUCTS; MALONDIALDEHYDE MODIFIED LOW-DENSITY LIPOPROTEIN; ATHEROSCLEROSIS; BIOMARKER

Introduction Oxidative modification of low-density lipoprotein (LDL) plays a crucial role in atherogenesis.1,2 Oxidized LDL (oxLDL), which is found not only in the arterial wall but also in the circulation, is associated with vascular pathology such as foam cell formation by macrophages and the inflammatory response in the vasculature.1,2

Malondialdehyde is a reactive aldehyde species formed from the breakdown of polyunsaturated lipids by reactive oxygen species. Malondialdehyde-modified LDL (MDA–LDL) is an oxidative modification of LDL that is a useful biomarker of oxidative stress.3 Glycation of biomolecules, such as lipids, proteins and lipoproteins, leads to the

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K Kotani, R Caccavello, N Taniguchi et al. sRAGE inversely correlated to oxidized LDL in asymptomatic subjects formation of heterogeneous products, termed advanced glycation end products (AGEs) in pathological, but also normoglycaemic, conditions.4 − 6 The engagement of AGEs with the receptor for AGEs (RAGE), which is expressed in the cells of the arterial wall as a truncated-type receptor composed of an extracellular ligand-binding domain without cytosolic and transmembrane domains, promotes the activation of nuclear factor-κB and inflammatory cytokines, as well as the induction of oxidative stress, leading to vascular damage.4 − 6 In this AGEs–RAGE system, RAGE has a C-truncated secretory isoform of the receptor protein, termed soluble RAGE (sRAGE), which is produced extracellularly in response to RAGE signalling.6 − 8 The sRAGE exhibits similar ligand binding specificity to RAGE; therefore, sRAGE competes with cell-bound RAGE for ligand binding, functioning as a ‘decoy’ receptor that abrogates cellular activation.6 − 8 Furthermore, sRAGE has been shown to exert antiatherogenic effects, and higher circulating levels of sRAGE can confer resistance to AGEmediated inflammatory pathways.6,8 Thus, sRAGE is considered to be a potentially important marker in atherogenesis and cardiovascular disease (CVD). A previous experimental study reported that oxLDL could be one of the RAGE ligands.9 The interaction of oxLDL with RAGE, in relation to sRAGE, is of great interest; however, there are no clinical data to show whether there is an association between circulating sRAGE and oxLDL levels. The present study investigated the clinical association between sRAGE and oxLDL in humans; MDA–LDL was measured as a biomarker of oxLDL.3

Subjects and methods STUDY POPULATION This

cross-sectional

study

recruited

community-dwelling participants from education classes within primary healthcare settings in Tochigi-Kyoto, Japan, between 2008 and 2009. Included subjects were required to be asymptomatic and not receiving any medication. The exclusion criteria were smoking on self-report and a history of cardio/cerebrovascular, thyroid, haematological, collagen or severe hepatic and renal diseases. The study was approved by the Ethics Committee of Jichi Medical University, Japan (No. A08-76) and all subjects gave their written informed consent prior to initiation of the study.

CLINICAL ASSESSMENTS Clinical data were obtained after an overnight fast. Calculation of body mass index (BMI) was based on each subject’s weight (kg) and height (m2) whilst wearing lightweight clothing and no shoes. Blood pressure (BP) was determined with a mercury sphygmomanometer on the subject’s right arm while the patient was seated; mean BP was calculated using the equation: diastolic BP + (systolic BP – diastolic BP) / 3. Fasting venous blood was collected into tubes containing 15 mM sodium fluoride for glucose measurement (mixed immediately) and tubes without anticoagulant for serum separation. The blood samples were centrifuged at 1600 g for 15 min at room temperature and stored at –80 °C until use. Serum LDL cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG) and plasma glucose concentrations were measured enzymatically using commercial kits (Kyowa Medex, Tokyo, Japan) according to the manufacturer’s instructions. The serum MDA–LDL level was measured by enzyme-linked immunosorbent assay (ELISA; Sekisui Co., Tokyo, Japan) with intra- and interassay coefficients of variation

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K Kotani, R Caccavello, N Taniguchi et al. sRAGE inversely correlated to oxidized LDL in asymptomatic subjects of 6.5% and 9.0%, respectively.3 Serum sRAGE was also measured by an ELISA (R&D Systems, Minneapolis, MN, USA) and the intra- and interassay coefficients of variation for this assay were 1.9% and 5.5%, respectively.10

STATISTICAL ANALYSES The mean ± SD or median (interquartile range) values were calculated. A simple linear correlation test (Pearson’s correlation coefficient) and stepwise multiple linear regression analysis on sRAGE (F for the entry set to 2) were used to observe the correlation between sRAGE and other measured variables including MDA–LDL. The TG values were log-transformed in these analyses because of their skewed distribution. Statistical significance was defined as P < 0.05 and analyses were carried out using the SPSS® statistical package, version 16.0 (SPSS Inc., Chicago, IL, USA).

Results The study included 33 subjects (15 males, 18 females). Mean ± SD/median (interquartile

range) values for the measured variables were: age 65 ± 4 years; BMI 23.4 ± 2.5 kg/m2; mean BP 97 ± 12 mmHg; LDL-C 3.46 ± 0.68 mmol/l; HDL-C 1.80 ± 0.40 mmol/l; TG 1.19 (0.91 − 1.44) mmol/l; glucose 5.29 ± 0.55 mmol/l; sRAGE 1101 ± 502 ng/l; and MDA– LDL 57.6 ± 17.4 IU/l. Correlation analyses for sRAGE with the other variables including MDA–LDL are presented in Table 1. Simple linear correlation analysis revealed a significant inverse correlation between sRAGE and BMI (P = 0.01) and between sRAGE and MDA– LDL (P = 0.04). Stepwise multiple linear regression analysis identified an independent and significant inverse correlation between sRAGE and BMI (P < 0.01) and between sRAGE and MDA–LDL (P = 0.03).

Discussion The present study demonstrated an independent, significant and inverse correlation between sRAGE and MDA–LDL, where MDA–LDL was used as a biomarker for oxLDL. This inverse relationship may be

TABLE 1: Correlation analyses of soluble receptor for advanced glycation end products (sRAGE) with other measured variables in asymptomatic subjects (n = 33) Linear correlation analysis Variable

r-value

Age Gender, males Body mass index Mean blood pressure LDL cholesterol HDL cholesterol Triglyceridesa Plasma glucose Malondialdehyde-LDL

0.16 −0.05 −0.43 0.12 −0.07 −0.07 −0.04 −0.15 −0.36

Stepwise multiple linear regression analysis

Statistical significance

b-value

Statistical significance

NS NS P = 0.01 NS NS NS NS NS P = 0.04

Not extracted Not extracted −0.43 Not extracted Not extracted Not extracted Not extracted Not extracted −0.36

– – P < 0.01 – – – – – P = 0.03

aTriglyceride

values were log-transformed because of their skewed distribution. LDL, low-density lipoprotein; HDL, high-density lipoprotein; NS, not statistically significant (P > 0.05).

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K Kotani, R Caccavello, N Taniguchi et al. sRAGE inversely correlated to oxidized LDL in asymptomatic subjects due to a putative antiatherosclerotic effect of sRAGE in quenching oxLDL particles, which have been experimentally documented as a RAGE ligand.9 Since sRAGE and oxLDL are both target markers in atherogenesis and CVD,1,2,6 and there are no reports of a correlation between these markers, the findings of the present study are important as they suggest a possible interaction between sRAGE and oxLDL. Lipid peroxidation may be linked to the glycation of LDL, and glycoxidative modification of LDL can include an AGE (Nε[carboxymethyl]lysine-protein adduct)-like epitope.11 A positive relationship between circulating AGEs and sRAGE levels has been reported in nondiabetic12 and diabetic patients.10,13 In contrast, the present study showed an inverse correlation between circulating oxLDL and sRAGE levels, suggesting that a different mechanism may be involved in AGE–RAGE interactions compared with oxLDL–RAGE interactions. One study reported an inverse relationship between levels of RAGE in monocytes and circulating sRAGE levels in diabetic patients,14 while another study reported an inverse relationship between urinary 8-iso-prostaglandin (PG) F2α, which is one of the biomarkers of lipid peroxidation, and circulating sRAGE levels in diabetic patients.15 The results of the present study agree with these previous studies, although the populations and oxidative stress-related markers are different,14,15 and supports the association between sRAGE and oxidative stress. There are several possible explanations regarding the mechanisms that might be responsible for the inverse relationship between circulating sRAGE and oxLDL. One theory is that an extra burden of RAGE ligands increases the binding and/or consumption/elimination of sRAGE resulting in lower levels of this decoy receptor. Since

oxLDL can be a RAGE ligand,9 increased production of oxLDL may consume/ eliminate sRAGE in subjects with increased oxidative stress/inflammation who are predisposed to atherosclerosis. Furthermore, other RAGE ligands may be present in the inflammatory and oxidative milieu where oxLDL occurs. These may include not only AGEs but also diverse ligands, such as proinflammatory cytokine-like mediators of the S100/calgranulin family, amphoterin, β2integrin Mac-1 on leukocytes, the high mobility group box chromosomal protein 1 and amyloid-β peptide.16,17 It seems reasonable to suggest that these RAGE ligands may concomitantly affect the binding of sRAGE–ligand complexes, including oxLDL. Another potential explanation could involve the lack of a compensatory increase of circulating sRAGE as part of an antiatherogenic process, perhaps mediated by genetic polymorphisms,18,19 which may enhance the oxidative milieu and lead to increased oxLDL formation. In fact, low circulating sRAGE levels have been reported to be associated with CVD outcomes, suggesting that individuals with originally low sRAGE levels may have an increased risk of atherosclerosis.5 It is uncertain whether the findings from the present study are the consequence of oxidative stress or a potential contributing factor. Thus, future studies are warranted to clarify the biological mechanisms responsible for the inverse relationship between sRAGE and oxLDL. The present study also demonstrated an independent, significant and inverse correlation between sRAGE and BMI, which has also been observed previously.8 Obesity is generally an inflammatory and oxidative condition20,21 that is closely and positively associated with RAGE ligands.17 Interestingly, it has been shown that RAGE is

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K Kotani, R Caccavello, N Taniguchi et al. sRAGE inversely correlated to oxidized LDL in asymptomatic subjects upregulated during the differentiation process to mature adipocytes, and adipocyterelated sRAGE production can, therefore, be reduced in obese individuals.22 This supports the idea that the inverse correlation between circulating sRAGE and BMI is, in part, due to the upregulation of RAGE. There were several limitations to the present study. The relatively small sample size and the cross-sectional design cannot confirm a causal relationship between sRAGE and MDA–LDL. The study population was restricted to relatively healthy subjects. The pathophysiological roles of sRAGE may differ between healthy and diseased states,14 so comparative studies with more varied populations should be investigated. In addition, other relevant variables, including AGEs and other RAGE ligands, were not

measured. These issues will be addressed in future studies. In summary, an independent, significant and inverse correlation was shown to exist between sRAGE and MDA–LDL in asymptomatic subjects. This inverse relationship may harbour an antiatherogenic effect of sRAGE as a modulator of oxLDL. More studies are needed to confirm these observations.

Acknowledgements This study was supported in part jointly by Jichi Medical University, Tochigi, Japan and by Touro University–California, Vallejo, USA.

Conflicts of interest The authors had no conflicts of interest to declare in relation to this article.

• Received for publication 7 April 2012 • Accepted subject to revision 10 April 2012 • Revised accepted 1 August 2012 © SAGE Publications Ltd 2012 References 1 Steinberg D: Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 1997; 272: 20963 – 20966. 2 Holvoet P: Relations between metabolic syndrome, oxidative stress and inflammation and cardiovascular disease. Verh K Acad Geneeskd Belg 2008; 70: 193 – 219. 3 Kondo A, Muranaka Y, Ohta I, et al: Relationship between triglyceride concentrations and LDL size evaluated by malondialdehyde-modified LDL. Clin Chem 2001; 47: 893 – 900. 4 Cerami A, Vlassara H, Brownlee M: Role of nonenzymatic glycosylation in atherogenesis. J Cell Biochem 1986; 30: 111 – 120. 5 Falcone C, Emanuele E, D’Angelo A, 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 – 1037. 6 Yan SF, Ramasamy R, Schmidt AM: Mechanisms of disease: advanced glycation end-products and their receptor in inflammation and diabetes complications. Nat Clin Pract Endocrinol Metab 2008; 4: 285 – 293. 7 Park IH, Yeon SI, Youn JH, et al: Expression of a novel secreted splice variant of the receptor for advanced glycation end products (RAGE) in

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18 Jang Y, Kim JY, Kang SM, et al: Association of the Gly82Ser polymorphism in the receptor for advanced glycation end products (RAGE) gene with circulating levels of soluble RAGE and inflammatory markers in nondiabetic and nonobese Koreans. Metabolism 2007; 56: 199 – 205. 19 Gaens KH, Ferreira I, van der Kallen CJ, et al: Association of polymorphism in the receptor for advanced glycation end products (RAGE) gene with circulating RAGE levels. J Clin Endocrinol Metab 2009; 94: 5174 – 5180. 20 Vincent HK, Taylor AG: Biomarkers and potential mechanisms of obesity-induced oxidant stress in humans. Int J Obes (Lond) 2006; 30: 400 – 418. 21 Lumeng CN, Saltiel AR: Inflammatory links between obesity and metabolic disease. J Clin Invest 2011; 121: 2111 – 2117. 22 Unoki H, Bujo H, Yamagishi S, et al: Advanced glycation end products attenuate cellular insulin sensitivity by increasing the generation of intracellular reactive oxygen species in adipocytes. Diabetes Res Clin Pract 2007; 76: 236 – 244.

Author’s address for correspondence: Dr Kazuhiko Kotani Department of Clinical Laboratory Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-City, Tochigi 329-0498, Japan. E-mail: [email protected]

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