Enhanced laminin carbonylation by monocytes in diabetes mellitus Elena Kostidou a , George Koliakos b, ⁎ , Daryoush Hamidi Alamdari b , Konstantinos Paletas c , Apostolos Tsapas c , Martha Kaloyianni a a Laboratory of Animal Physiology, School of Biology, Aristotle University, Thessaloniki 54124, Greece b Department of Biological Chemistry, Medical School, Aristotle University, Thessaloniki 54124, Greece c Laboratory for the Study of Metabolic Diseases, B’ Medical Clinic, Medical School, Aristotle University, Thessaloniki 54124, Greece Received 14 December 2006; received in revised form 2 March 2007; accepted 13 March 2007 Available online 28 March 2007 Abstract Objectives: Monocyte–extracellular matrix interactions have been implicated in atherosclerosis pathophysiology. In the present study we evaluated the oxidation of laminin by monocytes derived from either diabetic patients or healthy volunteers. Moreover, reactive oxygen species production was measured. Monocyte attachment and migration through oxidized and non-oxidized laminin were also studied. Design and methods: Laminin oxidation was tested by a sensitive ELISA assay in isolated monocytes. ROS production was measured with fluorescent indicators. 35 S-methionine was used for evaluating monocyte attachment. Monocyte migration through laminin was examined on transwells. Results: Monocytes derived from patients with diabetes mellitus showed an increased ability to carbonylate and attach to laminin. Diabetic monocytes produced increased levels of ROS as compared to controls. Our results showed the involvement of the alpha2 integrin subunit in monocyte attachment to both native and oxidized laminin in control and diabetic monocytes. Conclusions: The results indicate a modified interaction between monocytes and laminin in diabetes. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Monocytes; Laminin; Carbonylation; Reactive oxygen species (ROS); Attachment; Alpha2 integrin subunit; Migration; Diabetes mellitus Introduction Leukocytes, and in particular monocytes, are a key source of oxygen radicals in inflammatory states producing ROS such as superoxide anions, hydrogen peroxide and hydroxyl radicals. Leukocyte-derived ROS cause oxidative damage to several macromolecules like nucleic acids, lipids, or proteins. Reaction of free radicals with the side chains of lysine, arginine, proline and threonine residues of proteins leads to the formation of carbonyl derivatives [1]. Carbonyl derivatives can be also mediated by Michael addition of reactive carbonyls (RCS) to the nucleophilic sites of proteins, as well as from the oxidative cleavage of proteins [2]. Carbonylation may act as key factor in the development and progression of a variety of human diseases, such as diabetes and atherosclerosis [2–4]. As a result, protein carbonyl groups provide a reasonable marker for free radical-induced protein oxidation. Considerable evidence indicates that the maintenance of protein redox status is of great importance for cell function, whereas structural changes in proteins are considered to be among the molecular mechanisms leading to diabetic complica- tions [5]. Extracellular matrix (ECM) proteins are important for cell function. Moreover, since extracellular matrix proteins, especially in the sub-endothelial basement membranes, are exposed to ROS, their oxidation may lead to changes in the microenvironment and therefore may represent a novel mechanism in the onset of vascular complications in diabetic patients. Laminins are multidomain and multifunctional cross-shaped glycoproteins which are key structural and functional compo- nents of the basement membrane. Moreover, they play a central role in many biological functions such as cell adhesion, differentiation and migration of several cell types [6–8]. Recent studies indicated that post-translational modifications of the Clinical Biochemistry 40 (2007) 671 – 679 ⁎ Corresponding author. Fax: +30 231 099 9004. E-mail address: koliakos@med.auth.gr (G. Koliakos). 0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.03.011