RE S E A RC H
Serum Levels of Advanced Glycation End Products (AGEs) are Inversely Associated with the Number and Migratory Activity of Circulating Endothelial Progenitor Cells in Apparently Healthy Subjects So Ueda,1 Sho-ichi Yamagishi,2 Takanori Matsui,2 Yoshihiro Noda,1 Shin-ichiro Ueda,1 Yuko Jinnouchi,1 Ken-ichiro Sasaki,1 Masayoshi Takeuchi3 & Tsutomu Imaizumi1 1 Department of Internal Medicine, Division of Cardio-Vascular Medicine, Kurume, Japan 2 Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume, Japan 3 Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa, Japan
Keywords AGEs; Atherosclerosis; Diabetes; EPC. Correspondence Sho-ichi Yamagishi, M.D., Ph.D., Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830–0011, Japan. Tel.: (81) 942-31-7873; Fax: (81) 942-31-7873; E-mail:
[email protected]
doi: 10.1111/j.1755-5922.2011.00264.x
SUMMARY Objectives: Endothelial progenitor cells (EPCs) have been shown to participate in the process of vascular repair, thus playing a protective role against cardiovascular disease (CVD). It is known that atherosclerotic risk factors could affect EPC number and function. Advanced glycation end products (AGEs) contribute to the pathogenesis of atherosclerosis as well. However, as far as we know, there is no report to show the relationship between serum AGE levels and circulating EPCs in humans. Therefore, in this study, we investigated whether serum level of AGEs was associated with EPC number and functions in apparently healthy subjects, independent of traditional cardiovascular risk factors. Research Design and Methods: Apparently healthy volunteers (34.6 ± 6.9 years old, 40 males and 8 females) who were not on any medications underwent a complete history and physical examination, determination of blood chemistries, including AGEs, and number, differentiation and migratory activity of circulating EPCs. Results: Serum AGEs levels were 9.20 ± 1.85 U/mL. Multiple stepwise regression analysis revealed that serum levels of AGEs and smoking were independently correlated with reduced number of EPCs. Further, female, AGEs, and reduced HDL-cholesterol levels were independently associated with impaired migratory activity of circulating EPCs. Conclusions: This study demonstrated for the first time that the serum level of AGEs was one of the independent correlates of decreased cell number and impaired migratory activity of circulating EPCs in apparently healthy subjects. Our present observations suggest that even in young healthy subjects, serum level of AGEs may be a biomarker that could predict the progression of atherosclerosis and future cardiovascular events.
Abbreviations Endothelial progenitor cells (EPCs); cardiovascular disease (CVD); advanced glycation end products (AGEs); body weight (BW); body mass index (BMI); blood pressure (BP); high-density lipoprotein (HDL-C); low-density lipoprotein (LDL-C); glycosylated hemoglobin A1c (HbA1c); mononuclear cells (MNCs); enzyme-linked immunosorbent assay (ELISA); phycoerythrin (PE);
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monoclonal antibody (mAb); kinase-insert domain receptor (KDR); streptavidin fluorescein isothiocyanate (Stv-FITC); fluorescence-activated cell sorting (FACS); 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-AcLDL); stromal-derived factor (SDF); 4 ,6-diamidino-2-phenylindole (DAPI); standard deviation (SD).
Introduction Endothelial progenitor cells (EPCs) have been shown to participate in the process of vascular repair, contributing to the
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preservation of structurally and functionally intact endothelium [1,2]. Since impaired endothelial cell growth and function are an initial event of atherosclerosis [3,4], which could lead to the development and progression of cardiovascular disease (CVD), EPCs have been supposed to play a protective role against CVD via maintenance of endothelial integrity and function. Recently, EPCs were discovered in peripheral blood, and circulating EPC number and functions were inversely correlated with cardiovascular risk factors among apparently healthy people and in patients with CVD [1,5,6]. Further, decreased cell number and/or impaired activity of EPCs in peripheral blood were also associated with endothelial dysfunction, a surrogate marker of atherosclerosis [1,5–7]. These observations suggest that traditional cardiovascular risk factors could affect the number and properties of circulating EPCs; thus being involved in CVD. Reducing sugars can react nonenzymatically with amino groups of protein to form Amadori products. These early glycation products undergo further complex reaction such as rearrangement, dehydration, and condensation to become irreversibly cross-linked, heterogeneous fluorescent derivatives, termed advanced glycation end products (AGEs) [8–11]. The formation and accumulation of AGEs have been known to progress in a normal aging process and at an accelerated rate under hyperglycemic or oxidative stress conditions [8,9,12]. There is a growing body of evidence that AGEs contribute to the development and progression of atherosclerosis [13–15] and that serum level of AGEs could be a biomarker of endothelial dysfunction and future cardiovascular events in both diabetic and nondiabetic subjects [16–20]. However, as far as we know, there is no report to show the relationship between serum AGE levels and circulating EPCs in humans. Therefore, in this study, we investigated whether serum level of AGEs was associated with EPC number and functions in apparently healthy subjects, independent of traditional cardiovascular risk factors.
Subjects and Methods Subjects Apparently healthy volunteers (34.6 ± 6.9 years old, 40 males and 8 females) who were not on any medications were enrolled in this study. We excluded any patients with neoplastic disorders, active inflammatory diseases, CVD, renal diseases, and collagen diseases. The Ethical Committee of Kurume University approved this study. All participants gave informed consent.
Data Collection The medical history and use of smoking were ascertained by a questionnaire. Smoking was classified as current habitual use or not. Height and body weight (BW) were measured, and BMI (BMI: kilograms per meter squared) was calculated as an index of the presence or absence of obesity. Blood pressure (BP) was measured in the sitting position using an upright standard sphygmomanometer. Vigorous physical activity and smoking were avoided for at least 30 min before BP measurement. Blood was drawn from the antecubital vein in the morning after 12-h fast for determinants of lipids (HDL-cholesterol (HDLC), LDL-cholesterol (LDL-C), and triglycerides), plasma glucose,
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glycosylated hemoglobin (HbA1c), number of mononuclear cells (MNCs), and AGEs. Serum AGEs levels were measured with an enzyme-linked immunosorbent assay (ELISA) as described previously [21,22]. In this study, one unit (U) corresponds to 1 μg of glyceraldehyde-derived AGE-bovine serum albumin standard as described previously [22,23]. These samples were processed blindly. ELISA assay for AGEs was run in triplicate. Other blood chemistries were measured at a commercially available laboratory as described previously [21].
Flow Cytometric Analysis of EPCs MNCs were isolated from a total of 14 mL of peripheral blood by density gradient centrifugation with Ficoll (FicollPaque PLUS; GE Healthcare Bio-Sciences AB, Sweden). Then circulating EPCs were quantified with flow cytometric analysis as described previously [24,25]. In brief, MNCs were incubated with phycoerythrin-conjugated (PE-conjugated) anti-CD34 monoclonal antibody (mAb) (BD Biosciences Pharmingen, San Jose, CA, USA), PE-conjugated anti-CD133 mAb (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and biotin-conjugated antikinase insert domain receptor (KDR) mAb (NOVUS, Littleton, CO, USA), whereas streptavidin fluorescein isothiocyanateconjugated (Stv-FITC-conjugated) antimouse F(ab )2 (BD Biosciences Pharmingen) was used as a secondary antibody. Samples were analyzed by fluorescence-activated cell sorting (FACS) (Becton Dickinson, San Diego, CA, USA). CD34+ KDR+ or CD133+ KDR+ cells were quantified and expressed as the number of cells per total MNCs (%).
Differentiated EPCs MNCs were plated onto a 6-well tissue culture coated with fibronectin (Sigma, St. Louis, MO, USA) and maintained with endothelial basal medium containing 20% fetal bovine serum plus 0.1% human epidermal growth factor, 0.4% human fibroblast growth factor-B, 0.1% vascular endothelial growth factor, 0.1% ascorbic acid, 0.04% hydrocortisone, 0.1% insulinlike growth factor-1, 0.1% heparin, 0.1% gentamicin, and 0.1% amphotericin (Clonetics, San Diego, CA, USA). After 4 days in culture, nonadherent cells were removed by washing phosphatebuffered saline, and then adherent cells were incubated with 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-AcLDL) (Biomedical Technologies Inc., Stoughton, MA, USA) for 1 h. The cells were then fixed with 2% paraformaldehyde and incubated with FITClabeled lectin from Ulex europaeus (VECTOR Laboratories Inc., Burlingame, CA, USA). Dual-staining-positive cells for Dil-AcLDL and lectin were judged as differentiated EPCs [26]. Differentiated EPCs were expressed as the number of cells per MNCs (%).
Migrated EPCs After cultivating MNCs for 4 days, differentiated EPCs were detached as described previously [27]. Then 1 × 105 EPCs were resuspended in 500 μL endothelial basal medium and placed into the upper chamber of a modified-Boyden chamber (BD BioCoatTM Cell Cuture Inserts, BD Biosciences). The upper chamber was
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placed in a 24-well culture dish containing 500 μL endothelial basal medium supplemented with 20% fatal bovine serum and 100 ng/mL stromal cell-derived factor-1α (SDF-1α) (R&D Systems Inc., Minneapolis, MN, USA). After 24 h incubation at 37◦ C, transmigrated cells into the lower chamber were fixed with 2% paraformaldehyde and cell nuclei were counterstained with 4 , 6-diamidino-2-phenylindole (DAPI) (Dojindo Molecular Technologies Inc., Kumamoto, Japan). Migrated cells were counted in five random high-power fields under fluorescent microscope.
Statistical Methods Because of skewed distributions, the natural logarithmic (ln) transformations were performed for triglycerides. Data were expressed as mean ± standard deviation (SD). To compare the clinical parameters between male and female, we used the Student t-test or χ -square test. Smoking was coded as dummy variables. Univariate analysis was performed for correlates of cell number of CD34+ KDR+ , CD133+ KDR+ , differentiated or migratted EPCs. To determine independent correlates of these parameters, multiple stepwise logistic regression analysis was performed. Statistical significance was defined as P < 0.05. All statistical analyses were performed with the use of the SAS system (SAS Institute, Cary, NC, USA).
Univariate Parameters
β
SE
P value
Age Sex Height BW BMI Systolic BP Diastolic BP HR FPG HbA1c LDL-C HDL-C Triglyceridesa AGEs MNCs Smoking
−0.004 0.119 −0.005 −0.005 −0.017 −0.003 −0.003 −0.004 −0.007 −0.068 −0.002 0.003 −0.039 −0.051 0.000 −0.212
0.004 0.066 0.003 0.002 0.009 0.002 0.003 0.003 0.003 0.119 0.001 0.002 0.047 0.012 0.005 0.057
0.265 0.078 0.110 0.035 0.079 0.114 0.317 0.216 0.047 0.575 0.014 0.112 0.412
