Biology subjects

Clegg, P. D.

Publications and source records attributed to Clegg, P. D..

12 recordsLinked to original sources

bioRxiv2023

MskAge - An Epigenetic Biomarker of Musculoskeletal Age Derived from a Genetic Algorithm Islands Model

BackgroundAge is a significant risk factor for functional decline and disease of the musculoskeletal system, yet few biomarkers exist to facilitate ageing research in musculoskeletal tissues. Multivariate models based on DNA methylation, termed epigenetic clocks, have shown promise as markers of biological age. However, the accuracy of existing epigenetic clocks in musculoskeletal tissues are no more, and often less accurate than a randomly sampled baseline model. ResultsWe developed a highly accurate epigenetic clock, Msk-Age, that is specific to tissues and cells of the musculoskeletal system. MskAge was built using a penalised genetic algorithm islands model that addresses multi-tissue clock bias. The final model was trained on the transformed principal components of CpGs selected by the genetic algorithm, which are significantly enriched for pathways terms related to the skeletal system and mesenchyme development. We show that MskAge tracks epigenetic ageing ex vivo and in vitro. Epigenetic age estimates are rejuvenated to zero with cellular reprogramming and are accelerated at a rate of 0.45 years per population doubling. Remarkably, MskAge explains more variance associated with in vitro ageing of fibroblasts than the purpose-developed skin and blood clock. ConclusionThe precision of MskAge and its ability to capture perturbations in biological ageing make it a promising research tool for musculoskeletal and ageing biologists.

cell biology↗

bioRxiv2023

Mesenchymal Stromal Cell secretome is affected by tissue source, donor age and sex.

Variation in Mesenchymal Stromal Cell (MSC) function depending on their origin is problematic, as it may confound clinical outcomes of MSC therapy. Current evidence suggests that the therapeutic benefits of MSCs is primarily attributed to secretion of various biologically active factors (secretome). However, the effect of donor characteristics on the MSC secretome composition remains largely unknown. Here, we examined the influence of donor age, sex and tissue source, on the protein profile of the equine MSC secretome. Initially, we used dynamic metabolic labelling with stable isotopes combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify secreted proteins in MSC conditioned media (CM). Seventy proteins were classified as classically-secreted based on the rate of isotope label incorporation into newly synthesised proteins released into the extracellular space. Next, we analysed CM of bone marrow- (n = 14) and adipose-derived MSCs (n = 16) with label-free LC-MS/MS proteomics. Clustering analysis of 314 proteins detected across all samples identified tissue source as the main factor driving variability in MSC CM proteomes. Linear modelling applied to the subset of 70 secreted proteins identified tissue-related difference in the abundance of 23 proteins. There was an age-related decrease in the abundance of two proteins (CTHRC1, LOX), which has been validated with western blot and enzymatic activity assay. There was limited evidence of sex-related differences in protein abundance. In conclusion, this study provides evidence that tissue source and donor age contribute to heterogeneity in the protein composition of MSC secretomes which may influence the effects of MSC-based cell therapy. Significance statementThis research shows for the first time that donor age can influence the proteins secreted by mesenchymal stromal cells (MSC), which is considered the main mechanism through which they mediate their biological actions. This information may improve our understanding of the variable outcomes observed in MSC clinical studies, as well as identify optimal sources and donor selection for specific clinical applications.

cell biology↗

bioRxiv2023

The interfascicular matrix of energy storing tendons houses heterogenous cell populations disproportionately affected by ageing

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to injury, with incidence increasing with ageing. The interfascicular matrix (IFM), which binds tendon fascicles, plays a key role in energy storing tendon mechanics, and ageing alterations to the IFM negatively impact tendon function. While the mechanical role of the IFM in tendon function is well-established, the biological role of IFM-resident cell populations remains to be elucidated. Therefore, the aim of this study was to identify IFM-resident cell populations and establish how these populations are affected by ageing. Cells from young and old SDFTs were subjected to single cell RNA-sequencing, and immunolabelling for markers of each resulting population used to localise cell clusters. Eleven cell clusters were identified, including tenocytes, endothelial cells, mural cells and immune cells. One tenocyte cluster localised to the fascicular matrix, whereas nine clusters localised to the IFM. Interfascicular tenocytes and mural cells were preferentially affected by ageing, with differential expression of genes related to senescence, dysregulated proteostasis and inflammation. This is the first study to uncover the importance of the IFM niche for a diverse range of cell populations, and to identify age-related alterations specific to IFM-localised cells.

cell biology↗

bioRxiv2021

Structure-function Specialisation of the Interfascicular Matrix in the Human Achilles Tendon

ObjectiveTendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency; these tendons are prone to debilitating injuries, the incidence of which increases with ageing. In equine tendons, energy storage is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the positional anterior tibialis and energy storing Achilles tendons, testing the hypothesis that the Achilles IFM has specialised composition and mechanical properties, which are lost with ageing. MethodsWe used a multidisciplinary combination of mechanical testing, immunolocalisation and proteomics to investigate structure-function specialisations in functionally distinct human tendons and how these are altered with ageing. ResultsThe IFM in the energy storing Achilles tendon is more elastic and fatigue resistant than the IFM in the positional anterior tibialis tendon, with a trend towards decreased fatigue resistance with age in the Achilles IFM. With ageing, alterations occur predominantly to the proteome of the Achilles IFM. ConclusionThe Achilles tendon IFM is specialised for energy storage, and changes to its proteome with ageing are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of key energy storing specialisations and their changes with ageing offers insight towards developing effective treatments for tendinopathy. Key messagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIEnergy storing tendons in animals and humans are particularly prone to tendinopathy and the incidence increases with increasing age. C_LIO_LIPrevious work in some animal models has shown that the specialisation of tendon properties for energy storage is achieved primarily through adaptation of the interfascicular matrix, with specialisation lost in ageing. However, the structural specialisations that provide the human Achilles tendon with its energy storing ability, and how these are affected by ageing, remain to be established. C_LI What does this study add?O_LIWe demonstrate that the interfascicular matrix in the human Achilles tendon is specialised for energy storing, with increased elastic recoil and fatigue resistance, and that these specialisations are partially lost with ageing, likely due to alterations to the proteome of the interfascicular matrix. C_LI How might this impact on clinical practice or future developments?O_LIShort term, the specialist IFM mechanics we have demonstrated can be detected with new developments in ultrasound functional imaging, offering improved opportunities for contextual tendinopathy diagnostics. Personalised rehabilitation programmes can now be explored and designed specifically to target IFM mechanics. C_LIO_LILonger term, the knowledge of key specialisations in injury prone energy storing tendons and how they are affected by ageing, offers crucial insight towards developing cell or tissue engineering treatments targeted at restoring tendon structure and function post-injury, specifically targeted at the IFM. C_LI

bioengineering↗

bioRxiv2021

miR-24:Prdx6 interactions regulate oxidative stress and viability of myogenic progenitors during ageing

microRNAs regulate a myriad of physiological processes, including skeletal muscle regeneration and homeostasis. During ageing, changes in muscle fibre microenvironment contribute to the capability of satellite cells to regenerate the muscle in response to injury and loading stressors. In this study, we isolated murine satellite cells and primary myogenic progenitors from mice and humans to demonstrate that the microRNA miR-24-3p and its target peroxiredoxin 6 (Prdx6) play an important role in muscle regeneration during ageing, regulating satellite cell viability and their differentiation potential. Our results show upregulation of miR-24 during early stages of muscle regeneration in vivo in adult mice, suggesting a potential role of miR-24 at the early stages of muscle injury. On contrary, miR-24 was downregulated during regeneration of muscle of old mice. miR-24 was also downregulated, whereas its target gene Prdx6 was upregulated, in satellite cells isolated from old mice. miR-24 consistently regulated viability and myogenic potential of myogenic progenitors from both humans and old mice, suggesting that changes in miR-24 levels during ageing may contribute to defective early stages of muscle regeneration during ageing through affecting satellite cell viability and myogenic potential. This regulation likely occurs via miR-24 counteracting the generation of reactive oxygen species through Prdx6 de-repression in primary myogenic progenitors isolated from humans and old mice. We propose that downregulation of miR-24 in muscle of old mice following injury may be a protective mechanism against elevated ROS levels to maintain satellite cell viability and myogenic potential, acting through Prdx6 upregulation. However, as miR-24 is a regulator of p16 and p21, this downregulation may lead to increased satellite cell senescence, therefore representing an age-related failed compensatory mechanism.

cell biology↗

bioRxiv2020

Active synthesis of collagen (I) homotrimer and matrisomal proteins in Dupuytren's fibrosis

Dupuytrens disease is a common fibroproliferative disease of the palmar fascia of the hand with advanced cases treated surgically. Anti-tumour necrosis factor (TNF) injection has undergone phase 2 trials and may be effective in slowing early-stage disease progression. Here we sought to determine how new synthesis of type I collagen in Dupuytrens differs from normal palmar fascia samples and to analyse the role of TNF in aberrant collagen synthesis. Model non-fibrotic, but fibrous connective tissues, were used to analyse active type I collagen protein synthesis in development, ageing and degenerative disease, where it was restricted to early development and ruptured tissue. Dupuytrens tissue was shown to actively synthesise type I collagen, including abnormal type I collagen homotrimer. TNF- reduced COL1A2 gene expression only in the presence of serum in 2D cell culture and had opposing effects on collagen protein production in the presence or absence of serum. TNF- had only limited effects in 3D tendon-like constructs. Anti-TNF did not reduce type I collagen synthesis in 3D tendon-like constructs or prevent type I collagen homotrimer synthesis in Dupuytrens tissue. Hence, modulation of the TNF- pathway in Dupuytrens disease is unlikely to prevent the pathological collagen accumulation that is characteristic of fibrosis.

cell biology↗

bioRxiv2020

Collagen (I) homotrimer does not cause bone fragility but potentiates the osteogenesis imperfecta (oim) mutant allele

Type I collagen is the major structural component of bone where it exists as an (1)2(2)1 heterotrimer in all vertebrates. The osteogenesis imperfecta (oim) mouse model comprising solely homotrimeric (1)3 type I collagen, due to a dysfunctional 2 chain, has a brittle bone phenotype implying that the heterotrimeric form is required for physiological bone function. However, humans with rare null alleles preventing synthesis of the 2 chain have connective tissue and cardiovascular abnormalities (cardiac valvular Ehlers Danlos Syndrome), without evident bone fragility. Conversely a prevalent human single nucleotide polymorphism leading to increased homotrimer synthesis is associated with osteoporosis. Whilst the oim line is well-studied, whether homotrimeric type I collagen is functionally equivalent to the heterotrimeric form in bone has not been demonstrated. Col1a2 null and oim mouse lines were used in this study and bones analysed by microCT and 3-point bending. RNA was also extracted from heterozygote tissues and allelic discrimination analyses performed using qRT-PCR. Here we comprehensively show for the first time that mice lacking the 2(I) chain do not have impaired bone biomechanical or structural properties, unlike oim homozygous mice. However Mendelian inheritance was affected in male mice of both lines and male mice null for the 2 chain exhibited age-related loss of condition. The brittle bone phenotype of oim homozygotes could result from detrimental effects of the oim mutant allele, however, the phenotype of oim heterozygotes is known to be less severe. We used allelic discrimination to show that the oim mutant allele is not downregulated in heterozygotes. We then tested whether gene dosage was responsible for the less severe phenotype of oim heterozygotes by generating compound heterozygotes. Data showed that compound heterozygotes had impaired bone structural properties as compared to oim heterozygotes, albeit to a lesser extent than oim homozygotes. Hence, we concluded that the presence of heterotrimeric collagen-1 in oim heterozygotes alleviates the effect of the oim mutant allele but a genetic interaction between homotrimeric collagen-1 and the oim mutant allele leads to bone fragility.

physiology↗

bioRxiv2020

Metabolomic and Proteomic Stratification of Equine Osteoarthritis

Osteoarthritis (OA) is characterised by loss of articular cartilage, synovial membrane dysfunction and subchondral sclerosis. Few studies have used a global approach to stratify equine synovial fluid (SF) molecular profiles according to OA severity. SF was collected from 58 metacarpophalangeal (MCP) and metatarsophalangeal joints of racing Thoroughbred horses (Hong Kong Jockey Club; HKJC) and 83 MCP joints of mixed breed horses from an abattoir and equine hospital (biobank). Joints were histologically and macroscopically assessed for OA severity. For proteomic analysis, native SF and SF loaded onto ProteoMiner equalisation columns, to deplete high abundant proteins, were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and label-free quantification. Validation of selected differentially expressed proteins was undertaken using clinical SF collected during diagnostic investigations. Native SF metabolites were analysed using 1D 1H Nuclear Magnetic Resonance (NMR). 1,834 proteins and 40 metabolites were identified in equine SF. Afamin levels decreased with synovitis severity and four uncharacterised proteins decreased with OA severity. Gelsolin and lipoprotein binding protein decreased with OA severity and apolipoprotein A1 levels increased for mild and moderate OA. Within the biobank, glutamate levels decreased with OA severity and for the HKJC cohort, 2-aminobutyrate, alanine and creatine increased with severity. Proteomic and metabolomic integration was undertaken using linear regression via Lasso penalisation modelling, incorporating 29 variables (R2=0.82) with principal component 2 able to discriminate advanced OA from earlier stages, predominantly driven by H9GZQ9, F6ZR63 and alanine. Combining biobank and HKJC datasets, discriminant analysis of principal components modelling prediction was good for mild OA (90%). This study has stratified equine OA using both metabolomic and proteomic SF profiles and identified a panel of markers of interest which may be applicable to grading OA severity. This is also the first study to undertake computational integration of NMR metabolomic and LC-MS/MS proteomic datasets of any biological system.

molecular biology↗

bioRxiv2020

Equine synovial fluid small non-coding RNA signatures in early osteoarthritis

BackgroundOsteoarthritis remains one of the greatest causes of morbidity and mortality in the equine population. The inability to detect pre-clinical changes in osteoarthritis has been a significant impediment to the development of effective therapies against this disease. Synovial fluid represents a potential source of disease-specific small non-coding RNAs (sncRNAs) that could aid in the understanding of the pathogenesis of osteoarthritis. We hypothesised that early stages of osteoarthritis would alter the expression of sncRNAs, facilitating the understanding of the underlying pathogenesis and potentially provide early biomarkers. MethodsSmall RNA sequencing was performed using synovial fluid from the metacarpophalangeal joints of both control and early osteoarthritic non-Thoroughbred horses. A group of differentially expressed sncRNAs was selected for further validation through qRT-PCR using an independent cohort of synovial fluid samples from control and early osteoarthritic horses. Bioinformatic analysis was performed in order to identify putative targets of the differentially expressed microRNAs and to explore potential associations with specific biological processes. ResultsResults revealed 22 differentially expressed sncRNAs including 13 microRNAs; miR-10a, miR-223, let7a, miR-99a, miR-23b, miR-378, miR-143 (and six novel microRNAs), four small nuclear RNAs; U2, U5, U11, U12, three small nucleolar RNAs; U13, snoR38, snord96, and one small cajal body-specific RNA; scarna3. Five sncRNAs were validated; miR-223 was significantly reduced in early OA and miR-23b, let-7a-2, snord96A and snord13 were significantly upregulated. Significant cellular functions deduced by the differentially expressed microRNAs included apoptosis (P < 0.0003), necrosis (P < 0.0009), autophagy (P < 0.0007) and inflammation (P < 0.00001). A conservatively filtered list of 57 messenger RNA targets was obtained; the top biological processes associated were regulation of cell population proliferation (P < 0.000001), cellular response to chemical stimulus (P < 0.000001) and cell surface receptor signalling pathway (P < 0.000001). ConclusionsSynovial fluid sncRNAs can be used as molecular biomarkers for early disease in equine osteoarthritic joints. The biological processes they regulate may play an important role in understanding early osteoarthritis pathogenesis. Characterising these dynamic molecular changes could provide novel insights on the process and mechanism of early osteoarthritis development and is critical for the development of new therapeutic approaches.

molecular biology↗

bioRxiv2020

Postnatal mechanical loading drives adaptation of tissues primarily through modulation of the non-collagenous matrix

Mature connective tissues demonstrate highly specialised properties, remarkably adapted to meet their functional requirements. Tissue adaptation to environmental cues can occur throughout life and poor adaptation commonly results in injury. However, the temporal nature and drivers of functional adaptation remain undefined. Here, we explore functional adaptation and specialisation of mechanically loaded tissues using tendon; a simple aligned biological composite, in which the collagen (fibre phase) and surrounding predominantly non-collagenous matrix (matrix phase) can be interrogated independently. Using an equine model of late development, we report the first phase-specific analysis of biomechanical, structural and compositional changes seen in functional adaptation, demonstrating adaptation occurs postnatally, following mechanical loading, and is almost exclusively localised to the non-collagenous matrix phase. These novel data redefine adaptation in connective tissue, highlighting the fundamental importance of non-collagenous matrix and suggesting that regenerative medicine strategies should change focus from the fibrous to the non-collagenous matrix phase of tissue.

cell biology↗

bioRxiv2020

Ex-Vivo Equine Cartilage Explant Osteoarthritis Model - A Metabolomics and Proteomics Study

Osteoarthritis (OA) is an age-related degenerative musculoskeletal disease characterised by loss of articular cartilage, synovitis, abnormal bone proliferation and subchondral bone sclerosis. Underlying OA pathogenesis is yet to be fully elucidated with no OA specific biomarkers in clinical use. Ex-vivo equine cartilage explants (n=5) were incubated in TNF-/IL-1{beta} supplemented culture media for 8 days, with media removed and replaced at 2, 5 and 8 days. Acetonitrile metabolite extractions of 8 day cartilage explants and media samples at all time points underwent 1D 1H nuclear magnetic resonance metabolomic analysis with media samples also undergoing mass spectrometry proteomic analysis. Within the cartilage, metabolites glucose and lysine were elevated following TNF-/IL-1{beta} treatment whilst adenosine, alanine, betaine, creatine, myo-inositol and uridine levels decreased. Within the culture media, four, four and six differentially abundant metabolites and 154, 138 and 72 differentially abundant proteins, with > 2 fold change, were identified for 1-2 day, 3-5 day and 6-8 day time points respectively. Nine potential novel OA neopeptides were elevated in treated media. Our innovative study has identified differentially abundant metabolites, proteins and extracellular matrix derived neopeptides, providing insightful information on OA pathogenesis, enabling potential translation for clinical markers and possible new therapeutic targets.

cell biology↗

bioRxiv2020

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon

Maintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000-fold differences in protein turnover rates, and overall faster protein turnover within the glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. These data provide insights into the complexity of proteome dynamics in tendon, likely required to maintain tissue homeostasis.

biochemistry↗