Characterisation of Extracellular Matrix Cross-Linking in Systemic Sclerosis

Systemic sclerosis (SSc) is a complex connective tissue disease associated with a high mortality. As development of skin fibrosis in SSc patients leads to major organ-based complications, finding a treatment that resolves this condition can help prevent disease progression. A major obstacle in the discovery of new effective anti-fibrotic therapies is that the exact mechanisms that lead to fibrotic progression and irreversibility have not been fully elucidated. The fibrotic phenotype is associated with increased accumulation of extracellular matrix (ECM) in an organ that severely impedes its function. Excessive cross-linking of the ECM is a major factor causing its resistance to remodelling by rendering sites of proteolysis less accessible to matrix metalloproteinases. The discovery of new cross-linking sites during disease progression may therefore improve our molecular understanding of the resistance of the ECM to proteolytic breakdown, act as a clinical marker for resolvable vs non-resolvable fibrosis, and contribute to the development of new therapeutic approaches. Transglutaminases are a group of cross-linking enzymes that play a pivotal role in fibrotic progression. A mass spectrometry-based method for the identification of transglutaminase cross-linking sites from complex biological samples has been developed in this project. A model transglutaminase cross-linked peptide was synthesized and its fragmentation behaviour was analysed. After having found the optimal fragmentation conditions the analysis of cross-linking sites was automated using MassMatrix software. The resulting score values were utilised to identify true-positive matches. This technique was successfully applied to an analysis of the transglutaminase cross-linking site of purified fibrinogen dimers. In the final stages of the method development comparative studies of the ECM cross-linking pattern between control and pro-fibrotic dermal fibroblast cell culture and mouse models were performed. The limited number of new cross-linking sites found in the fibrotic models suggest that more significant changes occur at later fibrotic stages