M.J. Peffers

Absolute quantification of selected proteins in the human osteoarthritic secretome.

Osteoarthritis (OA) is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA.

Proteomic analysis reveals age-related changes in tendon matrix composition, with age-and injury-specific matrix fragmentation *

Background: Alterations in tendon matrix composition with aging and injury are poorly understood. Results: Aging and injury resulted in distinct protein profiles, with age-specific peptide fragmentation in injury. Conclusion: Identification of protein cleavages associated with aging and injury suggest impaired maintenance and repair in aged tendon. Significance: Novel peptide fragments identified are potential biomarkers of tendon injury and age-related degeneration. Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs from young and old horses using label-free relative quantification to identify differentially abundant proteins and peptide fragments between age groups. The protein profile of injured SDFTs from young and old horses was also assessed. The results demonstrate distinct proteomic profiles in young and old tendon, with alterations in the levels of proteins involved in matrix organization and regulation of cell tension. Furthermore, we identified several new peptide fragments (neopeptides) present in aged tendons, suggesting that there are age-specific cleavage patterns within the SDFT. Proteomic profile also differed between young and old injured tendon, with a greater number of neopeptides identified in young injured tendon. This study has increased the knowledge of molecular events associated with tendon aging and injury, suggesting that maintenance and repair of tendon tissue may be reduced in aged individuals and may help to explain why the risk of injury increases with aging.

Transcriptomic signatures in cartilage ageing.

IntroductionAge is an important factor in the development of osteoarthritis. Microarray studies provide insight into cartilage aging but do not reveal the full transcriptomic phenotype of chondrocytes such as small noncoding RNAs, pseudogenes, and microRNAs. RNA-Seq is a powerful technique for the interrogation of large numbers of transcripts including nonprotein coding RNAs. The aim of the study was to characterise molecular mechanisms associated with age-related changes in gene signatures.MethodsRNA for gene expression analysis using RNA-Seq and real-time PCR analysis was isolated from macroscopically normal cartilage of the metacarpophalangeal joints of eight horses; four young donors (4 years old) and four old donors (>15 years old). RNA sequence libraries were prepared following ribosomal RNA depletion and sequencing was undertaken using the Illumina HiSeq 2000 platform. Differentially expressed genes were defined using Benjamini-Hochberg false discovery rate correction with a generalised linear model likelihood ratio test (P < 0.05, expression ratios ± 1.4 log2 fold-change). Ingenuity pathway analysis enabled networks, functional analyses and canonical pathways from differentially expressed genes to be determined.ResultsIn total, the expression of 396 transcribed elements including mRNAs, small noncoding RNAs, pseudogenes, and a single microRNA was significantly different in old compared with young cartilage (± 1.4 log2 fold-change, P < 0.05). Of these, 93 were at higher levels in the older cartilage and 303 were at lower levels in the older cartilage. There was an over-representation of genes with reduced expression relating to extracellular matrix, degradative proteases, matrix synthetic enzymes, cytokines and growth factors in cartilage derived from older donors compared with young donors. In addition, there was a reduction in Wnt signalling in ageing cartilage.ConclusionThere was an age-related dysregulation of matrix, anabolic and catabolic cartilage factors. This study has increased our knowledge of transcriptional networks in cartilage ageing by providing a global view of the transcriptome.

Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471–39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24–48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

Transcriptome analysis of ageing in uninjured human Achilles tendon.

IntroductionThe risk of tendon injury and disease increases significantly with increasing age. The aim of the study was to characterise transcriptional changes in human Achilles tendon during the ageing process in order to identify molecular signatures that might contribute to age-related degeneration.MethodsRNA for gene expression analysis using RNA-Seq and quantitative real-time polymerase chain reaction analysis was isolated from young and old macroscopically normal human Achilles tendon. RNA sequence libraries were prepared following ribosomal RNA depletion, and sequencing was undertaken by using the Illumina HiSeq 2000 platform. Expression levels among genes were compared by using fragments per kilobase of exon per million fragments mapped. Differentially expressed genes were defined by using Benjamini-Hochberg false discovery rate approach (P <0.05, expression ratios 1.4 log2 fold change). Alternative splicing of exon variants were also examined by using Cufflinks. The functional significance of genes that showed differential expression between young and old tendon was determined by using ingenuity pathway analysis.ResultsIn total, the expression of 325 transcribed elements, including protein-coding transcripts and non-coding transcripts (small non-coding RNAs, pseudogenes, long non-coding RNAs and a single microRNA), was significantly different in old compared with young tendon (±1.4 log2 fold change, P <0.05). Of these, 191 were at higher levels in older tendon and 134 were at lower levels in older tendon. The top networks for genes differentially expressed with tendon age were from cellular function, cellular growth, and cellular cycling pathways. Notable differential transcriptome changes were also observed in alternative splicing patterns. Several of the top gene ontology terms identified in downregulated isoforms in old tendon related to collagen and post-translational modification of collagen.ConclusionsThis study demonstrates dynamic alterations in RNA with age at numerous genomic levels, indicating changes in the regulation of transcriptional networks. The results suggest that ageing is not primarily associated with loss of ability to synthesise matrix proteins and matrix-degrading enzymes. In addition, we have identified non-coding RNA genes and differentially expressed transcript isoforms of known matrix components with ageing which require further investigation.

Anatomical heterogeneity of tendon: Fascicular and interfascicular tendon compartments have distinct proteomic composition

Tendon is a simple aligned fibre composite, consisting of collagen-rich fascicles surrounded by a softer interfascicular matrix (IFM). The composition and interactions between these material phases are fundamental in ensuring tissue mechanics meet functional requirements. However the IFM is poorly defined, therefore tendon structure-function relationships are incompletely understood. We hypothesised that the IFM has a more complex proteome, with faster turnover than the fascicular matrix (FM). Using laser-capture microdissection and mass spectrometry, we demonstrate that the IFM contains more proteins, and that many proteins show differential abundance between matrix phases. The IFM contained more protein fragments (neopeptides), indicating greater matrix degradation in this compartment, which may act to maintain healthy tendon structure. Protein abundance did not alter with ageing, but neopeptide numbers decreased in the aged IFM, indicating decreased turnover which may contribute to age-related tendon injury. These data provide important insights into how differences in tendon composition and turnover contribute to tendon structure-function relationships and the effects of ageing.

Matrix assisted laser desorption ionization mass spectrometry imaging identifies markers of ageing and osteoarthritic cartilage.

IntroductionCartilage protein distribution and the changes that occur in cartilage ageing and disease are essential in understanding the process of cartilage ageing and age related diseases such as osteoarthritis. The aim of this study was to investigate the peptide profiles in ageing and osteoarthritic (OA) cartilage sections using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI).MethodsThe distribution of proteins in young, old and OA equine cartilage was compared following tryptic digestion of cartilage slices and MALDI-MSI undertaken with a MALDI SYNAPT™ HDMS system. Protein identification was undertaken using database searches following multivariate analysis. Peptide intensity differences between young, ageing and OA cartilage were imaged with Biomap software. Analysis of aggrecanase specific cleavage patterns of a crude cartilage proteoglycan extract were used to validate some of the differences in peptide intensity identified. Immunohistochemistry studies validated the differences in protein abundance.ResultsYoung, old and OA equine cartilage was discriminated based on their peptide signature using discriminant analysis. Proteins including aggrecan core protein, fibromodulin, and cartilage oligomeric matrix protein were identified and localised. Fibronectin peptides displayed a stronger intensity in OA cartilage. Age-specific protein markers for collectin-43 and cartilage oligomeric matrix protein were identified. In addition potential fibromodulin and biglycan peptides targeted for degradation in OA were detected.ConclusionsMALDI-MSI provided a novel platform to study cartilage ageing and disease enabling age and disease specific peptides in cartilage to be elucidated and spatially resolved.

Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading

Summary Objectives SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9. Methods Equine chondrocytes harvested from normal or OA joints were subjected to different osmotic loading patterns as either primary (P0) or passaged (P2) cells. The involvement of MEK–ERK signalling was demonstrated by using pharmacological inhibitors. In addition SOX9 gene stability was determined. Levels of transcripts encoding SOX9, Col2A1 and aggrecan were measured using qRT-PCR. De novo glycosaminoglycan synthesis of explants was determined with 35S sulphate during static hyperosmolar loading. Results MEK–ERK signalling increases glycosaminoglycans (GAG) synthesis in explants. Static hyperosmotic conditions significantly reduced SOX9 mRNA in normal P2 and OA P0 but not normal P0 chondrocytes. SOX9 mRNA was stabilised by hyperosmotic conditions. Cyclical loading of normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by MEK1/2 inhibition. Conclusions The response to osmotic loading of SOX9 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype. This variation may be important in disease progression.

Transcriptome-Wide Analysis of Messenger RNA Decay in Normal and Osteoarthritic Human Articular Chondrocytes

Messenger RNA (mRNA) decay rates control not only gene expression levels, but also responsiveness to altered transcriptional input. We undertook this study to examine transcriptome‐wide posttranscriptional regulation in both normal and osteoarthritic (OA) human articular chondrocytes.

Comprehensive protein profiling of synovial fluid in osteoarthritis following protein equalization

Summary Objective The aim of the study was to characterise the protein complement of synovial fluid (SF) in health and osteoarthritis (OA) using liquid chromatography mass spectrometry (LC-MS/MS) following peptide-based depletion of high abundance proteins. Design SF was used from nine normal and nine OA Thoroughbred horses. Samples were analysed with LC-MS/MS using a NanoAcquity™ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMiner™. Progenesis-QI™ LC-MS software was used for label-free quantification. In addition immunohistochemistry, western blotting and mRNA expression analysis was undertaken on selected joint tissues. Results The number of protein identifications was increased by 33% in the ProteoMiner™ treated SF compared to undepleted SF. A total of 764 proteins (462 with≥2 significant peptides) were identified in SF. A subset of 10 proteins were identified which were differentially expressed in OA SF. S100-A10, a calcium binding protein was upregulated in OA and validated with western blotting and immunohistochemistry. Several new OA specific peptide fragments (neopeptides) were identified. Conclusion The protein equalisation method compressed the dynamic range of the synovial proteins identifying the most comprehensive SF proteome to date. A number of proteins were identified for the first time in SF which may be involved in the pathogenesis of OA. We identified a distinct set of proteins and neopeptides that may act as potential biomarkers to distinguish between normal and OA joints.