INTRODUCTION: Osteoarthritis (OA) is a major chronic musculoskeletal disease, leading to pain and disability in affected people. Ageing is considered as one of the main factors leading to OA. Although mechanisms of the disease are still being investigated, it is known that cause of OA is not only mechanically driven, but rather alteration of molecular pathways introduce pathological changes in the articular cartilage. As the cartilage deterioration is principally initiated from the surface of the tissue, stratifying the anatomical regions of articular cartilage and investigating them separately would add a new level of knowledge to the subject. The articular cartilage comprises of three distinctive zones, the thinnest and most cellular superficial zone forms the surface of cartilage and chondrocytes in this zone appear flattened. Middle zone is located underneath it and provides an anatomic and functional bridge between the superficial and deep zones, chondrocytes in this zone distributed sparsely. The deep zone is responsible for providing the greatest resistance to compressive forces, here chondrocytes are arranged in columnar structure. We hypothesize that the cartilage proteome is affected by the zone, age and disease status. Therefore we subjected young healthy, old healthy and OA cartilage from the superficial, middle and deep zones of human articular knee cartilage to high mass resolution mass spectrometry and label-free quantification. METHODS: In this study we have divided donors into 3 groups: young (n=5, mean age=32), old (n=5, mean age = 71) and OA (n=5, mean age = 76). From each donor we collected superficial, middle and deep zones of knee articular cartilage using laser microdissection (LMD) technique (Figure. 1), in total giving us 9 groups. Human articular cartilage of OA patients derived from femoral side of the knee joint was obtained from Liverpool Musculoskeletal Biobank under Health Research Authority approval. Articular cartilage from the same side of the knee joint from donors of two different age and with no history of OA were obtained from Proteogenex Inc. with appropriate ethical approval. Cartilage samples were sectioned at 20µm thickness in coronal plane and collected onto PEN membrane slides (Leica). Different zones of articular cartilage were collected under a laser microdissection microscope LMD7000 (Leica) into solution of 25mM ammonium bicarbonate. Microdissected tissue was in-situ trypsin digested with the addition of RapiGest surfactant (Waters). Tryptic peptides from each samples were individually run using liquid chromatography-tandem mass spectrometry on an UltiMate 3000 Nano LC System coupled to a Q Exactive Quadrupole-Orbitrap instrument (Thermo Fisher). Progenesis™ QI for proteomics v4.0 was used for label-free quantification following protein identification using the Unihuman reviewed database in Mascot (Matrix Science), ANOVA values of p<0.05, fold change of >1.5 and at least 2 unique peptides for protein identification were determined significant. Pathway analysis of differential abundant proteins was performed in IPA (Qiagen). RESULTS SECTION: Using LMD an average for superficial zone 5mm2 of cartilage tissue was collected and for middle/deep zones 25mm2 area was collected. In total 514 proteins were identified in this study. Label-free quantification revealed differentially abundant proteins in groups by age, OA and zones (Table 1). Pair wise comparison of differentially abundant proteins in the superficial zone between young and old group demonstrated the ‘activation of inflammatory response’ (p=7.47x10-6),’ inflammation of joint’ (p=3.42x10-6) and ‘apoptosis’ (p=6.96x10-5) pathways with ageing. Comparison of old and OA groups in this zone showed the ‘activation of apoptosis’ (p=8.63x10-6) and ‘cell death’ (p=9.28x10-9) in the OA group, whereas ‘inflammatory response’ (p=1.35x10-4) was upregulated in the old group. Similarly to the superficial zone, the ‘inflammation of joint’ (p=1.1x10-5) pathway was activated in the deep zone of old samples in comparison to young group. However, the deep zone OA group demonstrated the ‘activation of inflammatory response’ (p=4.94x10-4) in comparison to the old group. Old group in contrast had ‘activation in cell death’ (p=4.93x10-9) pathway. DISCUSSION: In the current study we have demonstrated a difference in protein abundance dependent on zone within articular cartilage, and that during ageing and OA these proteins change. From the results of pathway analysis we suggest that the proteome in the superficial and deep zones in ageing are similar, as old group had activated state of inflammation pathway in these two zones. However, comparing old and OA groups there are major differences between superficial and deep zones. It seems that at the protein level in OA cartilage inflammation pathways were downregulated in the superficial zone but activated in the deep zone when compared to the old group. Moreover, cell death was predicted to be activated in the superficial zone of OA, however it was inhibited in the deep zone in comparison to the old group. This may be the indication that cellular death in the superficial zone during OA is the result of cartilage deterioration and the inability of chondrocytes to cope with the dramatic changes in the surrounding matrix. Whereas downregulation of this pathway in the deeper zone could be the outcome of chondrocyte clusters formation, where proliferation of chondrocytes occur. It was shown in animal model studies that changes in subchondral bone can occur prior changes in the articular cartilage. Hence, the higher level of inflammation in the deep zone of OA cartilage could be the sign of crosstalk between this zone and underlying subchondral bone due to close proximity. Activation of the inflammation pathway in the old group in comparison to young group could be due to inflammaging. SIGNIFICANCE/CLINICAL RELEVANCE: This study has demonstrated that by stratifying anatomical zones of articular cartilage the changes in molecular mechanisms can be investigated in more detail and possibly identifying new treatment targets and age or disease specific biomarkers. ACKNOWLEDGEMENTS: We would like to acknowledge Kazakhstan presidential scholarship “Bolashak” for funding this study. Mandy Peffers is funded by Wellcome Trust Clinical Intermediate fellowship.
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