Ishibashi et al. Cardiovascular Diabetology 2013, 12:125 CARDIO http://www.cardiab.com/content/12/1/125 VASCULAR DIABETOLOGY ORIGINAL INVESTIGATION Open Access Advanced glycation end products evoke endothelial cell damage by stimulating soluble dipeptidyl peptidase-4 production and its interaction with mannose 6-phosphate/insulin- like growth factor II receptor Yuji Ishibashi1, Takanori Matsui1, Sayaka Maeda1, Yuichiro Higashimoto2 and Sho-ichi Yamagishi1* Abstract Background: Advanced glycation end products (AGEs) and receptor RAGE interaction play a role in diabetic vascular complications. Inhibition of dipeptidyl peptidase-4 (DPP-4) is a potential therapeutic target for type 2 diabetes. However, the role of DPP-4 in AGE-induced endothelial cell (EC) damage remains unclear. Methods: In this study, we investigated the effects of DPP-4 on reactive oxygen species (ROS) generation and RAGE gene expression in ECs. We further examined whether an inhibitor of DPP-4, linagliptin inhibited AGE-induced soluble DPP-4 production, ROS generation, RAGE, intercellular adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) gene expression in ECs. Results: DPP-4 dose-dependently increased ROS generation and RAGE gene expression in ECs, which were prevented by linagliptin. Mannose 6-phosphate (M6P) and antibodies (Ab) raised against M6P/insulin-like growth factor II receptor (M6P/IGF-IIR) completely blocked the ROS generation in DPP-4-exposed ECs, whereas surface plasmon resonance revealed that DPP-4 bound to M6P/IGF-IIR at the dissociation constant of 3.59 x 10-5 M. AGEs or hydrogen peroxide increased soluble DPP-4 production by ECs, which was prevented by N-acetylcysteine, RAGE-Ab or linagliptin. Linagliptin significantly inhibited the AGE-induced ROS generation, RAGE, ICAM-1 and PAI-1 gene expression in ECs. Conclusions: The present study suggests that AGE-RAGE-induced ROS generation stimulates the release of DPP-4 from ECs, which could in turn act on ECs directly via the interaction with M6P/IGF-IIR, further potentiating the deleterious effects of AGEs. The blockade by linagliptin of positive feedback loop between AGE-RAGE axis and DPP-4 might be a novel therapeutic target for vascular injury in diabetes. Keywords: AGEs, RAGE, DPP-4, Mannose 6-phosphate/IGF-II receptor * Correspondence: [email protected] 1Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan Full list of author information is available at the end of the article © 2013 Ishibashi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ishibashi et al. Cardiovascular Diabetology 2013, 12:125 Page 2 of 9 http://www.cardiab.com/content/12/1/125 Background Methods The pathological role of the non-enzymatic modification Materials of amino groups of proteins, nucleic acids and lipids by An inhibitor of DPP-4, linagliptin was generously gifted reducing sugars such as glucose, a process that is also from Boehringer Ingelheim (Ingelheim, Germany). Bovine known as “Maillard reaction”, has become increasingly serum albumin (BSA) (essentially fatty acid free and evident in various types of diseases [1-3]. It is now well essentially globulin free, lyophilized powder), D-Mannose- established that early glycation products undergo further 6-phosphate (M6P) and N-acetylcysteine (NAC) were pur- progressive modification over time in vivo to the forma- chased from Sigma (St. Louis, MO, USA). D-glyceraldehyde tion of irreversibly cross-linked senescent macroprotein from Nakalai Tesque (Kyoto, Japan). Recombinant human derivatives termed “advanced glycation end products DPP-4 from R&D systems (Minneapolis, MN, USA). (AGEs)” [1-3]. The formation and accumulation of AGEs Hydrogen peroxide (H2O2) from Wako Pure Chemical In- in various tissues have been known to progress at a dustries Ltd. (Osaka, Japan). Antibody (Ab) directed against physiological aging and at an accelerated rate under human M6P/insulin-like growth factor II receptor (IGF- hyperglycemic conditions [1-3]. There is accumulating IIR) (M6P/IGF-IIR-Ab) and DPP-4 from Santa Cruz Bio- evidence that AGEs elicit oxidative stress generation and technology Inc. (Delaware, CA, USA). subsequently evoke inflammatory and thrombogenic re- actions in a variety of cells through the interaction with Cells the receptor for AGEs (RAGE), thereby being involved HUVECs were cultured in endothelial basal medium in vascular complications in diabetes [4-9]. supplemented with 2% fetal bovine serum, 0.4% bovine Dipeptidyl peptidase-4 (DPP-4), also known as CD26, is brain extracts, 10 ng/ml human epidermal growth factor a type II transmembrane glycoprotein expressed on vari- and 1 μg/ml hydrocortisone according to the supplier’s ous cell types with multifunctional properties [10,11]. instructions (Clonetics Corp., San Diego, CA). DPP-4 or DPP-4 not only plays a role in T cell activation and prolif- AGE treatment was carried out in a medium lacking epi- eration, but also modulates the physiological activity dermal growth factor and hydrocortisone. of many regulatory peptides, because it is involved in the cleavage of N-terminal amino acids from several Dihydroethidium (DHE) staining chemokines and neuropeptides [10,11]. Incretins such as HUVEC were treated with or without the indicated con- glucagon-like peptide-1 (GLP-1) and glucose-dependent centrations of DPP-4, 100 μg/ml AGE-BSA or 100 μg/ml insulinotropic polypeptides (GIP) are gut hormones se- non-glycated BSA in the presence or absence of 50 μM creted from L and K cells in the intestine in response to M6P, 5 μg/ml M6P/IGF-IIR-Ab, 10 nM or 0.5 μM food intake, respectively [12,13], both of which are target linagliptin for 4 hr, and then the cells were incubated proteins of DPP-4 and rapidly degraded and inactivated by with phenol red free Dulbecco's Modified Eagle Medium this proteolytic enzyme [10,11]. Since GLP-1 and GIP aug- containing 3 μM DHE (Molecular Probes Inc., Eugene, ment glucose-induced insulin release from pancreatic OR, USA). After 15 minutes, the cells were imaged b-cells, suppresses glucagon secretion, and slows gastric under a laser-scanning confocal microscope. Superoxide emptying [12,13], inhibition of DPP-4 has been proposed generation was evaluated by intensity of DHE staining. as a potential therapeutic target for the treatment of type The intensity was analyzed by microcomputer-assisted 2 diabetes. However, it remains unclear DPP-4 inhibition NIH image. could have beneficial effects on AGE-exposed endothelial cells (ECs). In other words, whether DPP-4 itself is in- Surface plasmon resonance (SPR) volved in vascular injury in diabetes remains unknown. Recombinant human IGF-IIR (100 μg/ml, R&D system) DPP-4 and D-Mannose-6-phosphate/insulin-like growth was immobilized via the amino groups to CM5 sensor factor II receptor (M6P/IGF-IIR) interaction contributes chip (GE Healthcare, Buckinghamshire, UK) with the aid to T cell activation [14]. Therefore, in this study, we first of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and investigated whether DPP-4 could directly act on human N-hydroxysuccinimide. For affinity measurements, the umbilical vein ECs (HUVECs) to stimulate reactive oxygen association and dissociation phases were monitored in a species (ROS) generation and RAGE gene induction via BIAcore 1000 (GE Healthcare). Recombinant human the interaction with M6P/IGF-IIR. We next examined the DPP-4 was injected into the flow cell at concentrations effects of AGEs on soluble DPP-4 production released of 0.1 and 0.3 μM at a flow rate of 10 μl/min at 25°C. from HUVECs. We further studied whether an inhibitor The sensor chip was regenerated with pulses of 20 mM of DPP-4, linagliptin inhibited the AGE-induced soluble Tris–HCl buffer (pH 8.0) containing 6 M urea to the DPP-4 production, ROS generation, RAGE, intercellular baseline level, followed by an extensive washing with the adhesion molecule-1 (ICAM-1) and plasminogen activator running buffer. Control experiments were performed inhibitor-1 (PAI-1) gene expression in HUVECs. with IGF-IIR-free channel on the same sensor chip. Ishibashi et al. Cardiovascular Diabetology 2013, 12:125 Page 3 of 9 http://www.cardiab.com/content/12/1/125 From the assay curves obtained, the control signals, re- Statistical analysis flecting the bulk effect of buffer, were subtracted using Unless otherwise indicated, all values were presented as BIA-evaluation 4.1 software (GE Healthcare). Equilib- means ± SE from at least 3 independent experiments. rium dissociation constant (KD) was determined using Statistical analyses were performed by one-way ANOVA the equation for 1:1 Langmuir binding. followed by the Scheffe F test for multiple comparisons, and p < 0.05 was considered statistically significant. All Real-time reverse transcription-polymerase chain statistical analyses were performed with the use of the reactions (RT-PCR) PASW Statistics system (version 18.0; IBM Corporation, HUVEC were treated with or without the indicated con- New York, NY, USA). centrations of DPP-4, 100 μg/ml AGE-BSA or 100 μg/ml non-glycated BSA in the presence or absence of 10 nM Results or 0.5 μM linagliptin for 4 hr. Then total RNA was We first examined the effects of DPP-4 on ROS gener- extracted with RNAqueous-4PCR kit (Ambion Inc., ation in HUVECs. As shown in Figure 1A and 1B, DPP- Austin, TX, USA) according to the manufacturer’sinstruc- 4 dose-dependently increased superoxide generation in tions. Quantitative real-time RT-PCR was performed using HUVECs; 500 ng/ml DPP-4-induced increase in ROS Assay-on-Demand and TaqMan 5 fluorogenic nuclease generation was completely blocked by the treatment chemistry (Applied Biosystems, Foster city, CA, USA) with 10 nM linagliptin, 50 μM M6P or 5 μg/ml M6P/ according to the supplier’srecommendation.IDsofprimers IGF-IIR-Ab.