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The incidence of cardiomyopathy and cardiac fibrosis is markedly increased in patients with diabetes mellitus. As cardiac fibrosis is mediated by myofibroblasts, we investigated the effect of diabetes-associated collagen glycation on the conversion of cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin (SMA) promoter, elevated levels of collagen I, α-SMA mRNA, and enhanced protein expression of α-SMA, ED-A fibronectin and cadherins. Increased expression of α-SMA was dependent on β1 integrins and on TGF-β. In collagen gel assays, MGO-collagen promoted faster contraction and cell migration was increased by MGO-collagen. In shear-force detachment assays, cells on MGO-collagen were less adherent, and β1 integrin activation and focal adhesion formation were inhibited. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of cardiac fibrosis in diabetes.

The incidence of cardiomyopathy and cardiac fibrosis is markedly increased in patients with diabetes mellitus. As cardiac fibrosis is mediated by myofibroblasts, we investigated the effect of diabetes-associated collagen glycation on the conversion of cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin (SMA) promoter, elevated levels of collagen I, α-SMA mRNA, and enhanced protein expression of α-SMA, ED-A fibronectin and cadherins. Increased expression of α-SMA was dependent on β1 integrins and on TGF-β. In collagen gel assays, MGO-collagen promoted faster contraction and cell migration was increased by MGO-collagen. In shear-force detachment assays, cells on MGO-collagen were less adherent, and β1 integrin activation and focal adhesion formation were inhibited. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of cardiac fibrosis in diabetes.

This thesis describes the study of the molecular packing and organisation of collagen molecules within a fibril. The first two chapters describe the background to the study. In Chapter 1, a review of the extracellular matrix concentrates on the structure and organisation of type I collagen. Chapter 2 summarises the theory of X-ray diffraction by fibres, and Chapter 3 describes X-ray sources and equipment used in data collection. Data treatments and data extraction methods (such as simulated annealing) are also discussed. Chapters4 and 5 present the results of the study. Chapter 4 describes the determination of the one-dimensional structure of type I collagen to 0.54 nm resolution using X-ray diffraction and isomorphous derivative phase determination. The significance of the electron density map is interpreted in light of the known amino acid sequence, showing possible variations in the nature of the helix pitch. More importantly, the conformations of the intermolecular crosslink forming non-helical telopeptides were determined. Chapter 5 provides a detailed background to the current understanding of the three dimensional packing structure of collagen, and presents the first model-independent phase determined structure of a natural fibre - the lateral packing structure of type I collagen in rat tail tendon. The data extraction methods described in Chapter 3 are employed to calculate an electron density map of anisotropic resolution, from which the 4 crosslink forming telopeptide segments within the quasi-hexagonal packing structure are identified. Conclusions are drawn concerning the nature of order/disorder within collagen fibrils and the validity of the compressed microfibril model of collagen molecular packing and organisation is discussed. Chapter 6 summaries the results and evaluates the success of the study. The potential for development of the techniques and results found for further studies are also discussed.