Protein and transcriptome signatures of cartilage ageing and disease

It is hypothesised that there are distinct mechanisms involved in cartilage ageing and disease which can be determined using next-generation technologies including mass spectrometry and RNA-sequencing. The aims of this thesis were to firstly characterise molecular mechanisms associated with age-related and arthritis-related changes in cartilage gene and protein signatures. Secondly the thesis developed new techniques to identify novel cleavage sites in matrix proteins and to quantify some known proteolytic events in articular cartilage using mass spectrometry-based proteomics platforms. Finally the levels of key proteinases and their inhibitors involved in the pathogenesis of OA were measured using mass spectrometry. Osteoarthritis (OA) is an extremely common cause of morbidity in both man and animals. OA involves the biomechanical failure of articular cartilage, together with changes in the subchondral bone and inflammation of the joints and leads to a variety of symptoms including pain, stiffness and reduced mobility. Age is an important factor in the development of OA and represents a huge challenge for society as whilst life span increases, the quality of life faced by an ageing population is often poor. Articular cartilage is susceptible to age-related diseases such as OA, but it is not an inevitable result of ageing and is a consequence of a complex inter-relationship between age and further predisposing factors. There have been major advances in technologies used to interrogate proteins and genes due to genome sequencing enabling gene and protein sequences to be determined. These ‘next-generation technologies’ include mass spectrometry (MS) and next-generation sequencing. This thesis has used these technologies in an attempt to address important questions relating to cartilage ageing and disease. The use of an inflammatory model of early OA in equine and human cartilage enabled the discovery and quantification of important proteins and pathways involved, using relative and absolute mass spectrometry techniques. In the equine secretome pathway enrichment analyses confirmed the up-regulation of glycolytic proteins. The novel proteins clathrin and LIM and SH3 domain protein-1 were identified for the first time in cartilage proteomics. QconCAT technology and gene expression analysis enabled normal and OA cartilage extract to be interrogated. Absolute quantification values were demonstrated for the first time for aggrecan; first and third globular domains, biglycan, cartilage oligomeric matrix protein, decorin and fibromodulin. Whilst a novel MS based technique enabled previously identified and novel extracellular matrix cleavage sites derived from matrix metalloproteinase 3 and a disintegrin and metalloproteinase with thrombospondin motifs 4 digestion of cartilage to be determined. Some of these sites of degradation were also evident in OA but not normal cartilage using matrix assisted laser desorption ionization imaging MS (MALDI-IMS). Tentative markers of OA and ageing cartilage were also demonstrated. Finally an RNA sequencing study on ageing equine cartilage found an age-related failure of matrix, anabolic and catabolic cartilage factors together with a reduction in Wnt signalling. This thesis developed novel proteomic methodologies to identify and quantify distinct differences between cartilage ageing and disease. Several proteins not previously described in cartilage were identified. In addition many novel cartilage degradation products were identified and age-related peptides were visualised in cartilage for the first time.