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Neal L. Millar - One of the best experts on this subject based on the ideXlab platform.
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single cell and spatial transcriptomics in human Tendon Disease indicate dysregulated immune homeostasis
Annals of the Rheumatic Diseases, 2021Co-Authors: Moeed Akbar, Lucy Macdonald, Lindsay A N Crowe, Iain B. Mcinnes, Konstantin Carlberg, Mariola Kurowskastolarska, Patrik L Stahl, S Snelling, Neal L. MillarAbstract:Tendinopathy; encompassing multifactorial Tendon disorders characterised by pain and functional limitation remains a significant burden in musculoskeletal medicine.1 Recent findings highlight a key role for immune mediated mechanisms in Tendon Disease supporting the concept that pivotal immunological and biomechanical factors conventionally associated with inflammatory rheumatic and musculoskeletal Diseases (RMDs) are manifest in Tendon.2 Single cell technologies3 (scRNAseq) are increasingly applied in rheumatology to identify key cellular phenotypes that drive Disease pathogenesis. Despite efforts with small cell numbers and heterogenous Tendon biopsies4 there remains no detailed spatial Tendon cell atlas to inform translational targeting. Herein, for the first time utilising scRNAseq and spatial transcriptomics (ST), we carry out cell–cell interaction analysis to build an atlas of the dynamic cellular environment that drives the development of chronic human Tendon Disease. In healthy (4 biopsies, n=3040 cells) and Diseased (5 biopsies, n=19 084 cells) Tendon we find a mix of endothelial, immune and stromal cells (figure 1A, online supplemental file 1). Each cell type group is present in Disease and healthy tissue but with distinct quantitative and qualitative characteristics. Within stromal populations we identified ‘mural type’ stromal cells (figure 1B). Mural cells, which include pericytes, are possible progenitor cells in Tendon5 and interestingly, these cells are phenotypically similar to NOTCH3 high mural cells described in rheumatoid arthritis (RA) synovium which can differentiate into fibroblasts following interactions with endothelial cells (ECs) via JAG1 .6 Cell–cell interaction and ST analysis indicate a similar phenomenon could occur within tendinopathy between mural cells and SEMA3G ECs (figure 1F). In all Diseased stromal cell populations, there was greater expression of genes for extracellular matrix proteins (eg, COL1A1 , COL3A1 , FN1 , BGN ) which is considered the hallmark feature of tendinopathy (online supplemental figure S2B). Furthermore, pathway analysis indicates stromal …
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Novel self-amplificatory loop between T cells and tenocytes as a driver of chronicity in Tendon Disease.
Annals of the rheumatic diseases, 2021Co-Authors: Emma Garcia-melchor, Giacomo Cafaro, Lucy Macdonald, Lindsay A N Crowe, Shatakshi Sood, Michael Mclean, Umberto G. Fazzi, Iain B. Mcinnes, Moeed Akbar, Neal L. MillarAbstract:Objectives Increasing evidence suggests that inflammatory mechanisms play a key role in chronic Tendon Disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and Tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence Disease perpetuation. Methods T cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged Tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes. Results Significant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio. Conclusions Interaction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human Tendon disorders.
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inflammatory mechanisms in tendinopathy towards translation
Nature Reviews Rheumatology, 2017Co-Authors: Neal L. Millar, George A C Murrell, Iain B. McinnesAbstract:Tendon disorders are common and confer a large socioeconomic burden. This Review discusses the role of inflammatory mechanisms in Tendon homeostasis and resolution of Tendon damage, which are crucial to consider in developing novel therapeutics for tendinopathies. Tendinopathy is a multifactorial spectrum of Tendon disorders that affects different anatomical sites and is characterized by activity-related Tendon pain. These disorders are common, account for a high proportion (∼30%) of referrals to musculoskeletal practitioners and confer a large socioeconomic burden of Disease. Our incomplete understanding of the mechanisms underpinning Tendon pathophysiology continues to hamper the development of targeted therapies, which have been successful in other areas of musculoskeletal medicine. Debate remains among clinicians about the role of an inflammatory process in tendinopathy owing to a lack of clinical correlation. The advent of modern molecular techniques has highlighted the presence of immune cells and inflammatory mechanisms throughout the spectrum of tendinopathy in both animal and human models of Disease. Key inflammatory mediators — such as cytokines, nitric oxide, prostaglandins and lipoxins — play crucial parts in modulating changes in the extracellular matrix within tendinopathy. Understanding the links between inflammatory mechanisms, Tendon homeostasis and resolution of Tendon damage will be crucial in developing novel therapeutics for human Tendon Disease.
Stephanie G Dakin - One of the best experts on this subject based on the ideXlab platform.
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Divergent roles of prostacyclin and PGE2 in human tendinopathy
BMC, 2019Co-Authors: Filip Bergqvist, Andrew J. Carr, Kim Wheway, Bridget Watkins, Udo Oppermann, Per-johan Jakobsson, Stephanie G DakinAbstract:Abstract Background Tendon Disease is a significant global healthcare burden whereby patients experience pain and disability; however, the mechanisms that underlie inflammation and pain are poorly understood. Herein, we investigated the role of prostaglandins as important mediators of inflammation and pain in tissues and cells derived from patients with tendinopathy. Methods We studied supraspinatus and Achilles Tendon biopsies from symptomatic patients with tendinopathy or rupture. Tendon-derived stromal cells (CD45negCD34neg) isolated from Tendons were cultured and treated with interleukin-1β (IL-1β) to investigate prostaglandin production. Results Diseased Tendon tissues showed increased expression of prostacyclin receptor (IP) and enzymes catalyzing the biosynthesis of prostaglandins, including cyclooxygenase-1 (COX-1), COX-2, prostacyclin synthase (PGIS), and microsomal prostaglandin E synthase-1 (mPGES-1). PGIS co-localized with cells expressing Podoplanin, a marker of stromal fibroblast activation, and the nociceptive neuromodulator NMDAR-1. Treatment with IL-1β induced release of the prostacyclin metabolite 6-keto PGF1α in Tendon cells isolated from Diseased supraspinatus and Achilles Tendons but not in cells from healthy comparator Tendons. The same treatment induced profound prostaglandin E2 (PGE2) release in Tendon cells derived from patients with supraspinatus Tendon tears. Incubation of IL-1β treated Diseased Tendon cells with selective mPGES-1 inhibitor Compound III, reduced PGE2, and simultaneously increased 6-keto PGF1α production. Conversely, COX blockade with naproxen or NS-398 inhibited both PGE2 and 6-keto PGF1α production. Tendon biopsies from patients in whom symptoms had resolved showed increased PTGIS compared to biopsies from patients with persistent tendinopathy. Conclusions Our results suggest that PGE2 sustains inflammation and pain while prostacyclin may have a protective role in human Tendon Disease
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chronic inflammation is a feature of achilles tendinopathy and rupture
British Journal of Sports Medicine, 2018Co-Authors: Stephanie G Dakin, J L Newton, Robert Hedley, Stephen Gwilym, Natasha Jones, Hamish Reid, Simon Wood, Graham Wells, Fernando O Martinez, Louise AppletonAbstract:Background Recent investigation of human tissue and cells from positional Tendons such as the rotator cuff has clarified the importance of inflammation in the development and progression of Tendon Disease. These mechanisms remain poorly understood in Disease of energy-storing Tendons such as the Achilles. Using tissue biopsies from patients, we investigated if inflammation is a feature of Achilles tendinopathy and rupture. Methods We studied Achilles Tendon biopsies from symptomatic patients with either mid-portion tendinopathy or rupture for evidence of abnormal inflammatory signatures. Tendon-derived stromal cells from healthy hamstring and Diseased Achilles were cultured to determine the effects of cytokine treatment on expression of inflammatory markers. Results Tendinopathic and ruptured Achilles highly expressed CD14+ and CD68+ cells and showed a complex inflammation signature, involving NF-κB, interferon and STAT-6 activation pathways. Interferon markers IRF1 and IRF5 were highly expressed in tendinopathic samples. Achilles ruptures showed increased PTGS2 and interleukin-8 expression. Tendinopathic and ruptured Achilles tissues expressed stromal fibroblast activation markers podoplanin and CD106. Tendon cells isolated from Diseased Achilles showed increased expression of pro-inflammatory and stromal fibroblast activation markers after cytokine stimulation compared with healthy hamstring Tendon cells. Conclusions Tissue and cells derived from tendinopathic and ruptured Achilles Tendons show evidence of chronic (non-resolving) inflammation. The energy-storing Achilles shares common cellular and molecular inflammatory mechanisms with functionally distinct rotator cuff positional Tendons. Differences seen in the profile of ruptured Achilles are likely to be attributable to a superimposed phase of acute inflammation and neo-vascularisation. Strategies that target chronic inflammation are of potential therapeutic benefit for patients with Achilles Tendon Disease.
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proteomic analysis of Tendon extracellular matrix reveals Disease stage specific fragmentation and differential cleavage of comp cartilage oligomeric matrix protein
Journal of Biological Chemistry, 2014Co-Authors: Stephanie G Dakin, R K W Smith, Dick Heinegard, Patrik Onnerfjord, A Khabut, Jayesh DudhiaAbstract:During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint Diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured Tendons during early and late Disease stages to identify Disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring Disease, we hypothesized that stimulation of Tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural Disease. Therefore, normal Tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured Tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to Disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from Tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage Tendon Disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for Tendon Disease.
Iain B. Mcinnes - One of the best experts on this subject based on the ideXlab platform.
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single cell and spatial transcriptomics in human Tendon Disease indicate dysregulated immune homeostasis
Annals of the Rheumatic Diseases, 2021Co-Authors: Moeed Akbar, Lucy Macdonald, Lindsay A N Crowe, Iain B. Mcinnes, Konstantin Carlberg, Mariola Kurowskastolarska, Patrik L Stahl, S Snelling, Neal L. MillarAbstract:Tendinopathy; encompassing multifactorial Tendon disorders characterised by pain and functional limitation remains a significant burden in musculoskeletal medicine.1 Recent findings highlight a key role for immune mediated mechanisms in Tendon Disease supporting the concept that pivotal immunological and biomechanical factors conventionally associated with inflammatory rheumatic and musculoskeletal Diseases (RMDs) are manifest in Tendon.2 Single cell technologies3 (scRNAseq) are increasingly applied in rheumatology to identify key cellular phenotypes that drive Disease pathogenesis. Despite efforts with small cell numbers and heterogenous Tendon biopsies4 there remains no detailed spatial Tendon cell atlas to inform translational targeting. Herein, for the first time utilising scRNAseq and spatial transcriptomics (ST), we carry out cell–cell interaction analysis to build an atlas of the dynamic cellular environment that drives the development of chronic human Tendon Disease. In healthy (4 biopsies, n=3040 cells) and Diseased (5 biopsies, n=19 084 cells) Tendon we find a mix of endothelial, immune and stromal cells (figure 1A, online supplemental file 1). Each cell type group is present in Disease and healthy tissue but with distinct quantitative and qualitative characteristics. Within stromal populations we identified ‘mural type’ stromal cells (figure 1B). Mural cells, which include pericytes, are possible progenitor cells in Tendon5 and interestingly, these cells are phenotypically similar to NOTCH3 high mural cells described in rheumatoid arthritis (RA) synovium which can differentiate into fibroblasts following interactions with endothelial cells (ECs) via JAG1 .6 Cell–cell interaction and ST analysis indicate a similar phenomenon could occur within tendinopathy between mural cells and SEMA3G ECs (figure 1F). In all Diseased stromal cell populations, there was greater expression of genes for extracellular matrix proteins (eg, COL1A1 , COL3A1 , FN1 , BGN ) which is considered the hallmark feature of tendinopathy (online supplemental figure S2B). Furthermore, pathway analysis indicates stromal …
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Novel self-amplificatory loop between T cells and tenocytes as a driver of chronicity in Tendon Disease.
Annals of the rheumatic diseases, 2021Co-Authors: Emma Garcia-melchor, Giacomo Cafaro, Lucy Macdonald, Lindsay A N Crowe, Shatakshi Sood, Michael Mclean, Umberto G. Fazzi, Iain B. Mcinnes, Moeed Akbar, Neal L. MillarAbstract:Objectives Increasing evidence suggests that inflammatory mechanisms play a key role in chronic Tendon Disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and Tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence Disease perpetuation. Methods T cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged Tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes. Results Significant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio. Conclusions Interaction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human Tendon disorders.
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inflammatory mechanisms in tendinopathy towards translation
Nature Reviews Rheumatology, 2017Co-Authors: Neal L. Millar, George A C Murrell, Iain B. McinnesAbstract:Tendon disorders are common and confer a large socioeconomic burden. This Review discusses the role of inflammatory mechanisms in Tendon homeostasis and resolution of Tendon damage, which are crucial to consider in developing novel therapeutics for tendinopathies. Tendinopathy is a multifactorial spectrum of Tendon disorders that affects different anatomical sites and is characterized by activity-related Tendon pain. These disorders are common, account for a high proportion (∼30%) of referrals to musculoskeletal practitioners and confer a large socioeconomic burden of Disease. Our incomplete understanding of the mechanisms underpinning Tendon pathophysiology continues to hamper the development of targeted therapies, which have been successful in other areas of musculoskeletal medicine. Debate remains among clinicians about the role of an inflammatory process in tendinopathy owing to a lack of clinical correlation. The advent of modern molecular techniques has highlighted the presence of immune cells and inflammatory mechanisms throughout the spectrum of tendinopathy in both animal and human models of Disease. Key inflammatory mediators — such as cytokines, nitric oxide, prostaglandins and lipoxins — play crucial parts in modulating changes in the extracellular matrix within tendinopathy. Understanding the links between inflammatory mechanisms, Tendon homeostasis and resolution of Tendon damage will be crucial in developing novel therapeutics for human Tendon Disease.
Zi Yin - One of the best experts on this subject based on the ideXlab platform.
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controlled release curcumin attenuates progression of Tendon ectopic calcification by regulating the differentiation of Tendon stem progenitor cells
Materials Science and Engineering: C, 2019Co-Authors: Yangwu Chen, Weiliang Shen, Xiao Chen, Yubin Xie, Mengfei Liu, Chenqi Tang, Jiayun Huang, Tian Qin, Weishan Chen, Zi YinAbstract:Tendon calcification is a common but intractable problem leading to pain and activity limitation when injury or tendinopathy progresses into the late stage. This is because Tendon stem/progenitor cells (TSPCs) can undergo aberrant osteogenic differentiation under inflammatory conditions. This study aims to investigate the effect of curcumin, a natural anti-inflammatory agent, on regulating the differentiation of TSPCs in Tendon calcification. With inflammatory stimulation, TSPCs showed higher alkaline phosphatase activity and more frequent formation of mineralized nodules which were verified in the culture system; however, curcumin significantly alleviated these pathological changes. In in vivo function analysis, chitosan microsphere-encapsulated curcumin was delivered to injured sites of rat Tendon ectopic calcification model. The inflammation in the Tendon tissues of the curcumin group was significantly relieved. Controlled-release curcumin partially rescued Tendon calcification and enhanced Tendon regeneration in animal model. This study demonstrates that controlled-release curcumin can manipulate the fate decision of TSPCs, and that it promotes the tenogenesis and inhibits the osteogenesis of TSPCs in a pathological microenvironment, which provides a possible new therapeutic strategy for Tendon Disease.
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single cell analysis reveals a nestin Tendon stem progenitor cell population with strong tenogenic potentiality
Science Advances, 2016Co-Authors: Zi Yin, Long Yang, Zefeng Zheng, Can Zhang, Weiliang Shen, Huanhuan Liu, Jialin Chen, Boon Chin Heng, Guoji Guo, Xiao ChenAbstract:The repair of injured Tendons remains a formidable clinical challenge because of our limited understanding of Tendon stem cells and the regulation of tenogenesis. With single-cell analysis to characterize the gene expression profiles of individual cells isolated from Tendon tissue, a subpopulation of nestin+ Tendon stem/progenitor cells (TSPCs) was identified within the Tendon cell population. Using Gene Expression Omnibus datasets and immunofluorescence assays, we found that nestin expression was activated at specific stages of Tendon development. Moreover, isolated nestin+ TSPCs exhibited superior tenogenic capacity compared to nestin- TSPCs. Knockdown of nestin expression in TSPCs suppressed their clonogenic capacity and reduced their tenogenic potential significantly both in vitro and in vivo. Hence, these findings provide new insights into the identification of subpopulations of TSPCs and illustrate the crucial roles of nestin in TSPC fate decisions and phenotype maintenance, which may assist in future therapeutic strategies to treat Tendon Disease.
R K W Smith - One of the best experts on this subject based on the ideXlab platform.
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autologous stem cells in achilles tendinopathy ascat protocol for a phase iia single centre proof of concept study
BMJ Open, 2018Co-Authors: A Goldberg, Razi Zaidi, Deirdre Brooking, Louise Kim, Michelle Korda, Lorenzo Masci, Ruth Green, Paul Odonnell, R K W SmithAbstract:Introduction Achilles tendinopathy (AT) is a cause of pain and disability affecting both athletes and sedentary individuals. More than 150 000 people in the UK every year suffer from AT. While there is much preclinical work on the use of stem cells in Tendon pathology, there is a scarcity of clinical data looking at the use of mesenchymal stem cells to treat Tendon Disease and there does not appear to be any studies of the use of autologous cultured mesenchymal stem cells (MSCs) for AT. Our hypothesis is that autologous culture expanded MSCs implanted into an area of mid-portion AT will lead to improved pain-free mechanical function. The current paper presents the protocol for a phase IIa clinical study. Methods and analysis The presented protocol is for a non-commercial, single-arm, open-label, phase IIa proof-of-concept study. The study will recruit 10 participants and will follow them up for 6 months. Included will be patients aged 18–70 years with chronic mid-portion AT who have failed at least 6 months of non-operative management. Participants will have a bone marrow aspirate collected from the posterior iliac crest under either local or general anaesthetic. MSCs will be isolated and expanded from the bone marrow. Four to 6 weeks after the harvest, participants will undergo implantation of the culture expanded MSCs under local anaesthetic and ultrasound guidance. The primary outcome will be safety as defined by the incidence rate of serious adverse reaction. The secondary outcomes will be efficacy as measured by patient-reported outcome measures and radiological outcome using ultrasound techniques. Ethics and dissemination The protocol has been approved by the National Research Ethics Service Committee (London, Harrow; reference 13/LO/1670). Trial findings will be disseminated through peer-reviewed publications and conference presentations. Trial registration number NCT02064062.
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proteomic analysis of Tendon extracellular matrix reveals Disease stage specific fragmentation and differential cleavage of comp cartilage oligomeric matrix protein
Journal of Biological Chemistry, 2014Co-Authors: Stephanie G Dakin, R K W Smith, Dick Heinegard, Patrik Onnerfjord, A Khabut, Jayesh DudhiaAbstract:During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint Diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured Tendons during early and late Disease stages to identify Disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring Disease, we hypothesized that stimulation of Tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural Disease. Therefore, normal Tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured Tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to Disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from Tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage Tendon Disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for Tendon Disease.
