Biology subjects

Clarke, E. J.

Publications and source records attributed to Clarke, E. J..

5 recordsLinked to original sources

bioRxiv2023

Proteome and phospholipidome interrelationship of synovial fluid-derived extracellular vesicles in equine osteoarthritis: An exploratory multi-omics study to identify composite biomarkers

Osteoarthritis causes progressive joint deterioration, severe morbidity, and reduced mobility in both humans and horses. Currently, osteoarthritis is diagnosed at late stages through clinical examination and radiographic imaging, hence it is challenging to address and provide timely therapeutic interventions to slow disease progression or ameliorate symptoms. Extracellular vesicles are cell-derived vesicles that play a key role in cell-to-cell communication and are potential sources for specific composite biomarker panel discovery. We here used a multi-omics strategy combining proteomics and phospholipidomics in an integral approach to identify composite biomarkers associated to purified extracellular vesicles from synovial fluid of healthy, mildly and severely osteoarthritic equine joints. Although the number of extracellular vesicles was unaffected by osteoarthritis, proteome profiling of extracellular vesicles by mass spectrometry identified 40 differentially expressed proteins (non-adjusted p<0.05) in osteoarthritic joints associated with 7 significant canonical pathways in osteoarthritis. Moreover, pathway analysis unveiled changes in disease and molecular functions during osteoarthritis development. Phospholipidome profiling by mass spectrometry showed a relative increase in sphingomyelin and a decrease in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine in extracellular vesicles derived from osteoarthritic joints compared to healthy joints. Unsupervised data integration revealed positive correlations between the proteome and the phospholipidome. Comprehensive analysis showed that some phospholipids and their related proteins increased as the severity of osteoarthritis progressed, while others decreased or remained stable. Altogether our data show interrelationships between synovial fluid extracellular vesicle-associated phospholipids and proteins responding to osteoarthritis pathology and which could be explored as potential composite diagnostic biomarkers of disease.

molecular biology↗

bioRxiv2023

Multi-omic temporal landscape of plasma and synovial fluid-derived extracellular vesicles using an experimental model of equine osteoarthritis

Extracellular vesicles contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Limited studies have profiled extracellular vesicles in osteoarthritic biofluids, such as plasma and synovial fluid. Due to the potential involvement in osteoarthritis pathogenesis, synovial fluid-derived extracellular vesicles have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of early osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived extracellular vesicle small non-coding RNA and protein cargo using small RNA sequencing and mass spectrometry proteomics. We explored the data with a multi-omic approach including time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling induced by temporal synovial fluid-derived extracellular vesicles derived from the model were analysed using luciferase-based transcription factor activity assays. Extracellular vesicle protein cargo appears to be more important during osteoarthritis progression than small non-coding RNA cargo. Cluster analysis revealed plasma-extracellular vesicles represented a time-dependant response to osteoarthritis induction, were principally derived from protein cargo and were associated with supramolecular complexes. Clusters for synovial fluid-derived extracellular vesicles were associated with an initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis revealed that plasma-derived extracellular vesicles correlated with day post induction and were primarily composed of proteins which may modulate lipid metabolism in osteoarthritis. Synovial fluid-derived extracellular vesicles significant factors represented intermediate filament and supramolecular complexes reflecting tissue repair responses to osteoarthritis induction. There was a significant interaction between time and osteoarthritis for cAMP response element, Nuclear factor-kappa B response element, serum response element and serum response factor response element reporters with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect. Local and systemic osteoarthritis-associated changes in extracellular vesicle cargo profiles in this in vivo model provided a unique opportunity to understand their role in disease propagation and progression and may represent novel biomarkers to stage osteoarthritis.

molecular biology↗

bioRxiv2022

Temporal Extracellular Vesicle Protein Changes following Intraarticular Treatment with Integrin α10β1-selected Mesenchymal Stem Cells in Equine Osteoarthritis

Equine osteoarthritis is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterised by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilises their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin 10{beta}1-selected mesenchymal stem cell treatment in an experimental model of equine osteoarthritis with longitudinal sampling. Adipose tissue derived, integrin 10-MSCs were injected into the osteoarthritis afflicted joint after 18 days post surgery. Sixty-nine synovial fluid samples were collected via aseptic arthrocentesis at day 0, 18, 21, 28, 35, and 70. Synovial fluid was hyaluronidase treated and extracellular vesicles isolated using differential ultracentrifugation. Extracellular vesicles were characterised using the Exoview human tetraspanin chip. Extracellular vesicle concentration, surface marker identification, fluorescent microscopy and tetraspanin colocalization analysis was undertaken, in conjunction with nanoparticle tracking analysis. For proteomics, extracellular vesicle pellets were suspended in urea lysis buffer. Samples were reduced, alkylated and digested on SP3 beads with trypsin/LysC. A data independent acquisition mode was utilised for nano liquid chromatography tandem mass spectrometry analysis on a Triple TOF 6600 mass spectrometer. A total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR[≤] 0.05) between control and osteoarthritis treated with MSCs.. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p=0.023), complement activation (classical pathway) (p=0.023), and collagen containing extracellular matrix (p=0.034). To date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis and an improved phenotype.

molecular biology↗

bioRxiv2022

Optical photothermal infrared spectroscopy can differentiate equine osteoarthritic plasma extracellular vesicles from healthy controls

BackgroundEquine osteoarthritis is a chronic degenerative disease of the articular joint, characterised by cartilage degradation resulting in pain and reduced mobility and thus is a prominent equine welfare concern. Diagnosis is usually at a late stage through radiographic examination, whilst treatment is symptomatic not curative. Extracellular vesicles are small nanoparticles that are involved in intercellular communication. The objective of this study was to investigate the feasibility of Raman and optical photothermal infrared spectroscopy to detect osteoarthritis using plasma-derived extracellular vesicles. MethodsPlasma samples were derived from thoroughbred racehorses. A total of 14 samples were selected (control; n= 6 and diseased; n=8). Extracellular vesicles were isolated using differential ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy, and human tetraspanin chips. Samples were then analysed using Raman and optical photothermal infrared spectroscopy. ResultsInfrared spectra were analysed between 950-1800 cm-1. Raman spectra had bands between the wavelengths of 900-1800 cm-1 analysed. Bands below 900 cm-1. Spectral data for both Raman and optical photothermal infrared spectroscopy was used to obtain a classification model and confusion matrices, characterising the techniques ability to distinguish diseased samples. Optical photothermal infrared spectroscopy could differentiate osteoarthritic extracellular vesicles from healthy with good classification (93.4%) whereas Raman displayed poor classification (64.3%). Plasma-derived extracellular vesicles from osteoarthritic horses contained increased signal for proteins, lipids and nucleic acids. Discussion/ conclusionFor the first time we demonstrated the ability to use optical photothermal infrared spectroscopy to interrogate extracellular vesicles and osteoarthritis-related samples. Optical photothermal infrared spectroscopy was superior to Raman in this study, and could distinguish osteoarthritis samples, suggestive of its potential use diagnostically to identify osteoarthritis in equine patients. This study demonstrates the potential of Raman and optical photothermal infrared spectroscopy to be used as a diagnostic tool in clinical practice, with the capacity to detect changes in extracellular vesicles from clinically derived samples.

molecular biology↗

bioRxiv2022

Small non-coding RNA landscape of extracellular vesicles from a post-traumatic model of equine osteoarthritis

Extracellular vesicles comprise an as yet inadequately investigated intercellular communication pathway in the field of early osteoarthritis. We hypothesised that small non-coding RNA expression pattern in synovial fluid and plasma would change during progression of experimental osteoarthritis. In this study, we used small RNA sequencing to provide a comprehensive overview of the temporal expression profiles of small non-coding transcripts carried by EVs derived from plasma and synovial fluid for the first time in a post-traumatic model of equine osteoarthritis. Additionally, we characterised synovial fluid and plasma-derived extracellular vesicles with respect to quantity, size, and surface markers. The differential expression of seven microRNAs in plasma and synovial fluid-derived extracellular vesicles; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a and four snoRNAs; U3, snord15, snord46, snord58 represent potential biomarkers for early OA. Bioinformatics analysis of the differentially expressed microRNAs in synovial fluid highlighted that in early OA these related to the inhibition of cell cycle, cell cycle progression, DNA damage and cell proliferation but increased cell viability, and differentiation of stem cells. Plasma and synovial fluid-derived extracellular vesicle small non-coding signatures have been established for the first time in a temporal model of osteoarthritis. These could serve as novel biomarkers for the evaluation of osteoarthritis progression or act as potential therapeutic targets.

molecular biology↗