Radiation Physics and Chemistry 71 (2004) 953–956 The structure and organisation of type-IV collagen in normal and glycated basement membrane Layla Ali a , D.A. Bradley a , R.E. Ellis a , E. Green a , J.G. Grossmann b , C.P. Winlove a, * a Biomedical Physics Group, School of Physics, University of Exeter, Exeter EX4 4QL, UK b Daresbury Laboratory, Synchrotron Radiation Department, CCLRC, Daresbury, Warrington WA4 4AD, UK 1. Introduction Basement membrane, a highly specialised type of extracellular matrix that displays complex structure, underlies the endothelium throughout the vascular system. It acts as a filter to the movement of water and solutes across the vessel wall, particularly in the kidney and throughout the microcirculation. Many of the vascular complications of diabetes have been attributed to deleterious changes in the function of the basement membrane arising from the glycation of its structural elements (Shinider and Khon, 1980), in particular the type-IV collagen that provides the frame- work of its structure. Yurcheno and Furthmayr (1984) have demonstrated lateral association of type-IV mole- cules in solution by rotary shadowing, while Timple et al. (1981) have suggested that aggregation of C-terminal globular regions of the tetramers results in the formation of a ‘chicken wire’ network of molecules. However, there would appear to be little evidence to support any of these models. The suggestion that type-IV collagen organises in the form of a network indicates the use of X-ray diffraction techniques as a means for determina- tion of its structure. Anterior lens capsule provides a thick, easily handled model system for study of the organization of type-IV collagen. Previous work by Gathercole et al. on stretched bovine lens capsule demonstrated notable features in the arrangement of type-IV collagen, including axial crystallinity and a form of liquid crystal behaviour. The aims of this project are to explore further the normal structure and organisation of type-IV collagen in basement membrane and for the first time to investigate changes that occur with glycation. The process of non-enzymatic glycation and its subsequent reaction of glycated proteins have been implicated as factors contributing to pathology in the lens, kidney, connective tissues and vasculature in diabetes (Shinider and Khon, 1980). 2. Experimental methodology Basement membrane constituting the lens capsule was collected from the eyes of horses in the age ranges, 0–5, 10–15 and 20–25 years, obtained fresh from a local abattoir. The lenses and lens capsule were dissected out and the anterior lens capsule peeled off as a disc, use subsequently being made only of the central part of the lens in order to avoid contributions in the diffraction pattern from zonular fibres. For some samples selective removal of proteoglycans from the basement membrane was undertaken. For this, lens capsules were extracted at 20  C for 24 h in 1 M NaCl, or phosphate buffered saline (PBS) containing heparinase at 5 unit/ml (Sigma) and 4 M guanidine hydrochloride (Fisher Biotech) or ex- posed to PBS containing 20 mM dithiothreitol (Fisher Biotech). For some of these samples the basement membranes were glycated by incubation in 50 mM glucose or 50mM ribose in PBS for up to 30 days at a temperature of 37  C. A number of samples were used in the unstrained (relaxed) condition while others were stretched under 100% humidity by uniaxial loading for a period of 48 h using the same technique applied elsewhere in stretching ARTICLE IN PRESS *Corresponding author. Tel.: +44-1392-264140; fax: +44- 1392-264111. E-mail address: c.p.winlove@exeter.ac.uk (C.P. Winlove). 0969-806X/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2004.05.024