Increased accumulation of the glycoxidation product Nε-(carboxymethyl)lysine in hearts of diabetic patients: generation and characterisation of a monoclonal anti-CML antibody

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Abstract

Heart failure is a condition closely linked to diabetes. Hyperglycaemia amplifies the generation of a major advanced glycation end product Nε-(carboxymethyl)lysine (CML), which has been associated with the development of vascular and inflammatory complications. An increased accumulation of CML in hearts of diabetic patients may be one of the mechanisms related to the high risk of heart failure. Therefore, we investigated the localization of CML in diabetic hearts.

To investigate the presence and accumulation of CML in tissues, a monoclonal anti-CML antibody was generated and characterised. With this novel monoclonal antibody against CML, the localization of CML was investigated by immunohistochemistry, in heart tissue of controls (n=9) and heart tissue of diabetic patients (n=8) without signs of inflammation or infarction. In addition, in the same subjects we studied the presence of CML in renal and lung tissues. CML staining was approximately sixfold higher in hearts from diabetic patients as compared to control hearts (2.0±0.3 and 0.3±0.2 A.U., respectively, P<0.01). CML deposition was localized in the small intramyocardial arteries in endothelial cells and smooth muscle cells, but not in cardiomyocytes. These arteries did not show morphological abnormalities. The intensity of staining between arteries at the epicardial, midcardial and endocardial side did not vary significantly within patients. In renal tissues, CML staining was most prominent in tubules and in atherosclerotic vessels, without differences in intensity between controls and diabetic patients. In non-infected lungs, no CML was detected.

In conclusion, CML adducts are abundantly present in small intramyocardial arteries in the heart tissue of diabetic patients. The accumulation of CML in diabetic hearts may contribute to the increased risk of heart failure in hyperglycaemia.

Introduction

Diabetes is associated with an increased risk of heart failure [1], [2]. Poor glycaemic control is independently associated with the incidence of hospitalisation and/or death due to heart failure among adult patients with diabetes [2], [3], [4]. Although a variety of mechanisms have been proposed, it is presently thought that the increased risk of heart failure with hyperglycaemia may be causally related to both micro- and macroangiopathy [3]. Posttranslational chemical modifications of proteins and lipoproteins are believed to play a role in the pathogenesis of micro- and macroangiopathy. In diabetic patients, advanced glycation end products (AGEs) form an important family of chemical modified proteins associated with glycaemic control [5]. AGEs have been implicated as causal factors in endothelial dysfunction associated with vascular diseases. They accumulate during aging and at an accelerated rate in diabetes [6], [7]. Although AGEs were originally characterised by their yellow-brown fluorescent colour and their ability to form cross-links with and between amino groups [7], the term AGEs is now used for a broad range of advanced products of the Maillard reaction including pentosidine, Nε-(carboxyethyl)lysine (CEL) and Nε-(carboxymethyl)lysine (CML). CML-modified polypeptides is a major group of AGEs present in vivo [8], [9]. CML is colourless, does not show fluorescence, nor does it occur as a cross-link in or between proteins [10]. CML can be formed on proteins by an oxidative cleavage of the Amadori product fructose-lysine [11], [12], and by a reaction of proteins with the peroxidation products of polyunsaturated fatty acids [13] or the dicarbonyl compound glyoxal [14], [15]. Furthermore, recent data indicated that myeloperoxidase activity may also be an important source of CML in tissue proteins [16]. Because of the requirement of glycation and oxidation in the formation of CML, CML is designated to be a glycoxidation product [17]. Immunohistochemical studies demonstrated enhanced accumulation of CML in vascular tissue from diabetic patients and, consistent with the concept that both glycation and oxidative stress are involved in the generation of CML, in atherosclerotic lesions in human subjects [18], [19]. In diabetic patients, the degree of CML accumulation has been correlated with the extent of complications [20], [21].

It was reported that CML adducts are ligands for the receptor of AGE (RAGE), thereby activating key cell signalling pathways such as NFκB activation with subsequent modulation of gene expression including the expression of VCAM-1 on endothelial cells and the induction chemotaxis of mononuclear phagocytes [21], [22], [23], [24], [25]. These findings link CML via a CML–RAGE interaction to the development of accelerated vascular complications [26], [27].

CML may thus contribute to angiopathy in diabetic hearts. Since the presence of CML in the heart tissue of diabetes has not been studied, we assessed the presence of CML in diabetic hearts using a novel monoclonal antibody against CML-modified proteins.

Section snippets

Reagents

Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pyrogen-free human serum albumin (HSA) was obtained from the Central Laboratory of Blood Transfusion (CLB, Amsterdam, the Netherlands). Methylglyoxal, 3-deoxyglucosone and pentosidine were prepared as described [28].

Preparation of CML-modified proteins

CML-human serum albumin (CML-HSA) and CML-keyhole limpet hemocyanin (CML-KLH) were prepared and characterized as described previously [9] with minor modifications. Briefly, 20-mg

Characterization of CML antibodies

Antisera against CML-modified KLH were raised in three mice and the mouse with the highest titer for CML-HSA was used for the production of monoclonal antibodies. Two out of forty positive clones, which belonged to the IgG1 class, were characterized in detail with comparable results. The characterization of one clone is presented. When HSA was incubated with increasing concentrations of glyoxylic acid (20–300 mM) and NaCNBH3 (0.125–2 M), there was a progressive increase in the number of CML

Discussion

Glycoxidation has been linked to the pathogenesis of vascular complications. We demonstrated, with the use of a novel monoclonal antibody against CML-modified proteins, that the glycoxidation product CML accumulates in heart tissue in patients with diabetes.

The first part of this study describes the development, characterization and use of a monoclonal antibody recognizing CML. The findings that the antibody reactivity, as determined in a ELISA, was proportional to CML, but not to CEL, as

Acknowledgements

This work was supported by a fellowship from the Diabetes Fonds Nederland (CGS) and a University Stimulating Fund grant of the Vrije Universiteit (CGS and VvH). Dr. H. Niessen is a recipient of the Dr. E. Dekker program of the Netherlands Heart Foundation (D99025).

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    This article is dedicated to my teacher Prof. Dr. Henk van den Bosch. He was of inestimable value for my scientific career. I owed him a great debt of gratitude.

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