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      Calcification of the Medial Layer of the Internal Thoracic Artery in Diabetic Patients: Relevance of Glycoxidation

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          Objective: The aim of this study was to evaluate the role of glycoxidation in the calcification of the internal thoracic artery (ITA) in diabetes mellitus (DM). Methods: ITA samples were obtained from 17 patients with type 2 DM (age 62.9 ± 10.5 years) and 12 age-matched, nondiabetic patients (age 62.5 ± 10.2 years) who underwent coronary artery bypass grafting. These samples were analyzed histopathologically and assessed for calcification by von Kossa staining and for glycoxidation by immunohistochemistry using anti-N<sup>Ε</sup>-(carboxymethyl)lysine (CML) antibody. Morphometric evaluation of calcification of the medial layer, intimal thickness and intima-to-media ratio was performed using NIH image software. To evaluate the mechanism of the interaction between calcification and glycoxidation, we developed an in vitro model of calcification of collagen that was chemically modified by glucose, glutaraldehyde or epoxy compound. The calcium-binding activity of these collagens was determined in hydrolysates using atomic absorption spectrophotometry. Results: ITAs of both diabetic and nondiabetic patients were free of atherosclerosis, and no differences were found between the two groups with regard to intimal thickness and intima-to-media ratio. Areas of calcification were noticed in both groups in the tunica media, but not in the tunica intima. Calcium deposits were localized within the extracellular matrix, which was immunohistochemically positive for CML. The extent of medial layer calcification was significantly greater in diabetic patients than nondiabetic subjects, but was independent of known risk factors such as hypertension, hyperlipidemia, obesity and history of old myocardial infarction. The binding activity of collagen was time-dependently increased with in vitro incubation of glucose. A significant increase in the calcium-binding ability was observed in glucose- and glutaraldehyde-modified collagens, but not in epoxy compound-modified collagen. Conclusion: Our results suggest that glycoxidative modification of the extracellular matrix, in particular collagen, of the vascular wall may enhance the development of ITA calcification in diabetic patients.

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          Most cited references 9

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          N (epsilon)-(carboxymethyl)lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction.

          Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGE). Recent immunological studies have suggested the potential role of AGE in atherosclerosis, aging, and diabetic complications. We previously prepared a monoclonal (6D12) as well as a polyclonal anti-AGE antibody and proposed the presence of a common AGE structure(s) that may act as a major immunochemical epitope [Horiuchi, S., Araki, N., & Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332]. The purpose of the present study was to determine the major epitope. Amino acid analysis of AGE-proteins indicated that N(epsilon)-(carboxymethyl)lysine (CML) was a major modified lysine residue. Immunologic studies demonstrated the positive reaction of 6D12 not only to all CML-modified proteins tested, but also to BSA modified with several aldehydes known to generate a CML-protein adduct, and a linear correlation between the CML contents of CML-BSA and their immunoreactivity to 6D12 up to approximately 8 mol/mol of protein. Further experiments with CML analogs revealed that the epitope of 6D12 is a CML-protein adduct with an important carbonyl group. In contrast to 6D12, our polyclonal anti-AGE antibody showed a significant but much weaker immunoreactivity to CML-BSA, suggesting that the polyclonal antibody contains two populations, one reactive to CML (CML-PA) and the other unreactive to CML (Non-CML-PA). Non-CML-PA separated from CML-PA by CML-BSA affinity chromatography did not react with all CML-modified preparations, but retained its property to react commonly with AGE preparations obtained from proteins, lysine derivatives, and monoaminocarboxylic acids. Therefore, it is clear that a CML-protein adduct is a major immunological epitope in AGE structures, but there still exist other major epitope(s) expressed commonly in AGE-proteins.
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            Recent advances in multifactorial regulation of vascular calcification.

            Calcification presents important clinical implications in cardiovascular diseases, especially in coronary arteries. Epidemiological evidence has shown the coexistence of vascular calcification with both atherosclerosis and osteoporosis, and increasing evidence has shown the role of hyperlipidemia and atherogenic phospholipids in vascular calcification. The etiology of vascular calcification is also increasingly recognized as an active process. Vascular calcification initiates with matrix vesicle formation and mineralization following a process similar to that in bone. In addition, many bone regulatory factors have been shown to be present in calcified atherosclerotic lesions. In this review, we focus on the new developments emerging during the past year in regulation of vascular calcification. Regulatory factors include matrix GLA protein, the phosphate cotransporter Pit-1, a calcium-sensing receptor related factor, osteoprotegerin, leptin, bisphosphonates and oxidized lipids. Some of these, including oxidized lipids, osteoprotegerin, and bisphosphonates, appear to regulate mineralization in both bone and vasculature and may account for the co-existence of osteoporosis and atherosclerotic calcification that is independent of age.
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              Advanced glycation endproducts accelerate calcification in microvascular pericytes.

              Vascular calcification in advanced atherosclerosis is frequently associated with diabetes, and is a predictor of future cardiovascular events. To investigate the molecular mechanisms of vascular calcification, we examined whether advanced glycation endproducts (AGE) formed at an accelerated rate under diabetes induce the osteoblastic differentiation of pericytes, a mesenchymal progenitor. First, von Kossa staining demonstrated that AGE significantly increased the number of calcified nodules in a bovine pericyte culture. AGE were also found to induce calcium accumulation in the pericyte monolayer in time- and dose-dependent manners. Second, quantitative reverse transcription-polymerase chain reaction revealed that AGE increased the pericyte levels of mRNAs coding for alkaline phosphatase and osteopontin, the representative markers for early and late osteoblastic differentiation, respectively. Alkaline phosphatase activity was actually enhanced by AGE. The results suggest that AGE have the ability to induce the osteoblatic differentiation of pericytes, which would contribute to the development of vascular calcification in diabetes. Copyright 1999 Academic Press.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                December 2003
                29 January 2004
                : 40
                : 6
                : 567-574
                aDepartment of Pathology, bSecond Department of Internal Medicine, cDepartment of Cardiovascular Surgery, School of Medicine, Fukuoka University, Fukuoka, dDepartment of Biochemistry, Kumamoto University School of Medicine, Kumamoto, Japan
                75807 J Vasc Res 2003;40:567–574
                © 2003 S. Karger AG, Basel

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                Page count
                Figures: 6, Tables: 2, References: 30, Pages: 8
                Research Paper


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