Computational Analysis of miR-140 and miR-135 as Potential Targets to Develop Combinatorial Therapeutics for Degenerative Tendinopathy.

Background: Degenerative tendinopathy, a condition causing movement restriction due to high pain, highly impacts productivity and quality of life. The healing process is a complex phenomenon and involves a series of intra-cellular and inter-cellular processes. Proliferation and differentiation of the tenocyte is a major and essential process to heal degenerative tendinopathy. The recent development in microRNA (miRNA)-mediated reprogramming of the cellular function through specific pathways opened door for the development of new regenerative therapeutics. Based on information about gene expression and regulation of tendon injury and healing, we attempted to evaluate the combinatorial effect of selected miRNAs for better healing of degenerative tendinopathy. Methods: The present study was designed to evaluate the combinatorial effect of two miRNAs (has-miR-140 and has-miR-135) in the healing process of the tendon. Publicly available information/data were retrieved from appropriate platforms such as PubMed. Only molecular data, directly associated with tendinopathies, including genes/proteins and miRNAs, were used in this study. The miRNAs involved in tendinopathy were analyzed by a Bioinformatics tools (e.g., TargetScan, miRDB, and the RNA22v2). Interactive involvement of the miRNAs with key proteins involved in tendinopathy was predicted by the Insilco approach. Results: Based on information available in the public domain, tendon healing-associated miRNAs were predicted to explore their therapeutic potentials. Based on computation analysis, focusing on the potential regulatory effect on tendon healing, the miR-135 and miR-140 were selected for this study. These miRNAs were found as key players in tendon healing through Rho-associated coiled-coil containing protein kinase 1 (ROCK1), IGF-1/PI3K/Akt, PIN, and Wnt signaling pathways. It was also predicted that these miRNAs may reprogram the cells to induce proliferation and differentiation activity. Many miRNAs are likely to regulate genes important for the tendinopathy healing process, and the result of this study allows an approach for miRNA-mediated regeneration of the tenocyte for tendon healing. Based on computational analysis, the role of these miRNAs in different pathways was established, and the results provided insights into the combinatorial approach of miRNA-mediated cell reprogramming. Conclusions: In this study, the association between miRNAs and the disease was evaluated to correlate the tendinopathy genes and the relevant role of different miRNAs in their regulation. Through this study, it was established that the synergistic effect of more than one miRNA on directed reprogramming of the cell could be helpful in the regeneration of damaged tissue. It is anticipated that this study will be helpful for the design of miRNA cocktails for the orchestration of cellular reprogramming events.

Integrative single-cell RNA and ATAC sequencing reveals that the FOXO1-PRDX2-TNF axis regulates tendinopathy.

Introduction: Tendinopathy, the most common form of chronic tendon disorder, leads to persistent tendon pain and loss of function. Profiling the heterogeneous cellular composition in the tendon microenvironment helps to elucidate rational molecular mechanisms of tendinopathy. Methods and results: In this study, through a multi-modal analysis, a single-cell RNA- and ATAC-seq integrated tendinopathy landscape was generated for the first time. We found that a specific cell subpopulation with low PRDX2 expression exhibited a higher level of inflammation, lower proliferation and migration ability, which not only promoted tendon injury but also led to microenvironment deterioration. Mechanistically, a motif enrichment analysis of chromatin accessibility showed that FOXO1 was an upstream regulator of PRDX2 transcription, and we confirmed that functional blockade of FOXO1 activity induced PRDX2 silencing. The TNF signaling pathway was significantly activated in the PRDX2-low group, and TNF inhibition effectively restored diseased cell degradation. Discussion: We revealed an essential role of diseased cells in tendinopathy and proposed the FOXO1-PRDX2-TNF axis is a potential regulatory mechanism for the treatment of tendinopathy.

Pro-inflammatory activity of long noncoding RNA FOXD2-AS1 in Achilles tendinopathy.

Achilles tendinopathy is a prevalent clinical problem that plagues athletes and general populations. Achilles tendon healing is a complex process, and so far, there is no successful long-term solution to Achilles tendinopathy in the field of microsurgery due to its poor natural regeneration ability. Limitations in understanding the pathogenesis of Achilles tendon development and Achilles tendon injury hinder clinical treatment developments. There is an increasing demand for innovative conservative treatments that can improve Achilles tendon injury. In this study, a Sprague-Dawley rat model of Achilles tendinopathy was established. Lentiviral vectors that interfere with the expression of FOXD2-AS1, miR-21-3p, or PTEN were injected every 3 days. Rats were euthanized after 3 weeks, and the effect of FOXD2-AS1, miR-21-3p, or PTEN on Achilles tendon healing was analyzed by histological observation, biomechanical test, and examinations of inflammatory factors and tendon markers. As measured, downregulating FOXD2-AS1 or upregulating miR-21-3p improved histological structure, suppressed inflammation, promoted the expression of tendon markers, and optimized the biomechanical properties of Achilles tendon. Upregulating PTEN was capable of reversing the promoting effect of inhibition of FOXD2-AS1 on Achilles tendon healing. As concluded, deficiency of FOXD2-AS1 accelerates the healing of Achilles tendon injury and improves tendon degeneration by regulating the miR-21-3p/PTEN axis and promoting the activation of the PI3K/AKT signaling pathway.

The Role of Epigenomics in Mapping Potential Precursors for Foot and Ankle Tendinopathy: A Systematic Review.

Tendinopathy of the foot and ankle is a common clinical problem for which the exact etiology is poorly understood. The field of epigenetics has been a recent focus of this investigation. The purpose of this article was to review the genomic advances in foot and ankle tendinopathy that could potentially be used to stratify disease risk and create preventative or therapeutic agents. A multi-database search of PubMed, Cochrane, Google Scholar, and clinicaltrials.gov from January 1, 2000 to July 1, 2022 was performed. A total of 18 articles met inclusion and exclusion criteria for this review. The majority of such research utilized case-control candidate gene association to identify different genetic risk factors associated with chronic tendinopathy. Polymorphisms in collagen genes COL5A1, COL27A1, and COL1A1 were noted at a significantly higher frequency in Achilles tendinopathy versus control groups. Other allelic variations that were observed at an increased incidence in Achilles tendinopathy were TNC and CASP8. The extracellular matrix (ECM) demonstrated macroscopic changes in Achilles tendinopathy, including an increase in aggrecan and biglycan mRNA expression, and increased expression of multiple matrix metalloproteinases. Cytokine expression was also influenced in pathology and aberrantly demonstrated dynamic response to mechanical load. The pathologic accumulation of ECM proteins and cytokine expression alters the adaptive response normal tendon has to physiologic stress, further propagating the risk for tendinopathy. By identifying and understanding the epigenetic mediators that lead to tendinopathy, therapeutic agents can be developed to target the exact underlying etiology and minimize side effects.Level of Evidence: Level IV: Systematic Review of Level II-IV Studies.

Scleraxis and collagen I expression increase following pilot isometric loading experiments in a rodent model of patellar tendinopathy.

The effect of mechanical load on tendinopathic tissue is usually studied in the context of identifying mechanisms responsible for tendon degradation. However, loading is also one of the most common treatments for tendinopathy. It is therefore possible that different loads result in different cellular responses within a tendon. To test this hypothesis, we first established a rodent model of tendinopathy that has a transcriptional signature similar to human tendinopathy. Tendinopathy was modeled in the rat by producing a lesion in the central core of the patellar tendon using a biopsy punch, followed by two weeks to allow scar formation. We performed 3' Tag RNA-Seq to identify genes that were differentially expressed between the native and scarred rat patellar tendon. Genes involved in extracellular matrix (ECM) structure and turnover were increased, mitochondrial genes were decreased, and there was no inflammatory signature in the tendinopathic tissue. These transcriptional changes phenocopy previously published whole transcriptome analysis in human tendinopathy. After validating the model, the initial response to injury and loading was determined. Two weeks after creation of the patellar tendon lesion, the tendon was loaded using either 4 × 30s isometric or a time-under-tension matched (360 × 0.33s) dynamic protocol. Injured +/- loading and contralateral control tendons were collected eighteen hours after loading, RNA was extracted, and gene expression was quantified using qRT-PCR of the scar with or without loading. The expression of scleraxis and type I collagen increased following isometric loading relative to those loaded dynamically. By contrast, the expression of type II collagen increased in the dynamic samples relative to those loaded isometrically. These data suggest that dynamic loading of a central core tendon injury increases fibrocartilage markers, whereas long isometric loads stimulate markers of tendon regeneration.

Screening of key biomarkers of tendinopathy based on bioinformatics and machine learning algorithms.

Tendinopathy is a complex multifaceted tendinopathy often associated with overuse and with its high prevalence resulting in significant health care costs. At present, the pathogenesis and effective treatment of tendinopathy are still not sufficiently elucidated. The purpose of this research is to intensely explore the genes, functional pathways, and immune infiltration characteristics of the occurrence and development of tendinopathy. The gene expression profile of GSE106292, GSE26051 and GSE167226 are downloaded from GEO (NCBI comprehensive gene expression database) and analyzed by WGCNA software bag using R software, GSE26051, GSE167226 data set is combined to screen the differential gene analysis. We subsequently performed gene enrichment analysis of Gene Ontology (GO) and "Kyoto Encyclopedia of Genes and Genomes" (KEGG), and immune cell infiltration analysis. By constructing the LASSO regression model, Support vector machine (SVM-REF) and Gaussian mixture model (GMMs) algorithms are used to screen, to identify early diagnostic genes. We have obtained a total of 171 DEGs through WGCNA analysis and differentially expressed genes (DEGs) screening. By GO and KEGG enrichment analysis, it is found that these dysregulated genes were related to mTOR, HIF-1, MAPK, NF-κB and VEGF signaling pathways. Immune infiltration analysis showed that M1 macrophages, activated mast cells and activated NK cells had infiltration significance. After analysis of THE LASSO SVM-REF and GMMs algorithms, we found that the gene MACROD1 may be a gene for early diagnosis. We identified the potential of tendon disease early diagnosis way and immune gene regulation MACROD1 key infiltration characteristics based on comprehensive bioinformatics analysis. These hub genes and functional pathways may as early biomarkers of tendon injuries and molecular therapy level target is used to guide drug and basic research.

Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo.

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon.

Identification of Differentially Expressed Genes Associated with Extracellular Matrix Degradation and Inflammatory Regulation in Calcific Tendinopathy Using RNA Sequencing.

Calcific tendinopathy (CT), developed due to calcium hydroxyapatite deposition in the rotator cuff tendon, mostly affects women in their 40 s and 50 s and causes severe shoulder pain. However, the molecular basis of its pathogenesis and appropriate treatment methods are largely unknown. In this study, we identified 202 differentially expressed genes (DEGs) between calcific and adjacent normal tendon tissues of rotator cuff using RNA sequencing-based transcriptome analysis. The DEGs were highly enriched in extracellular matrix (ECM) degradation and inflammation-related processes. Further, matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 13 (MMP13), two of the enzymes associated with ECM degradation, were found to be highly upregulated 25.85- and 19.40-fold, respectively, in the calcific tendon tissues compared to the adjacent normal tendon tissues. Histopathological analyses indicated collagen degradation and macrophage infiltration at the sites of calcific deposit in the rotator cuff tendon. Our study acts as a foundation that may help in better understanding of the pathogenesis associated with CT, and thus in better management of the disease.

Enhanced tendon restoration effects of anti-inflammatory, lactoferrin-immobilized, heparin-polymeric nanoparticles in an Achilles tendinitis rat model.

Gradual wear and tear can cause a local inflammatory response in tendons. The trauma and inflammatory reaction eventually impair the biomechanical properties of the tendon. In this study, we prepared lactoferrin-immobilized, heparin-anchored, poly(lactic-co-glycolic acid) nanoparticles (LF/Hep-PLGA NPs) and evaluated their in vitro anti-inflammatory effects on interleukin-1ß (IL-1ß)-treated tenocytes and in vivo tendon healing effects in a rat model of Achilles tendinitis. Long-term LF-deliverable NPs (LF/Hep-PLGA NPs) remarkably decreased mRNA levels of pro-inflammatory factors [cyclooxygenase-2 (COX-2), IL-1ß, matrix metalloproteinase-3 (MMP-3), MMP-13, IL-6, and tumor necrosis factor-α (TNF-α)] and increased mRNA levels of anti-inflammatory cytokines (IL-4 and IL-10) in both IL-1ß-treated tenocytes and the Achilles tendons of a collagenase-induced Achilles tendinitis rat model. Interestingly, anti-inflammatory LF/Hep-PLGA NPs greatly enhanced collagen content, mRNA levels of tenogenic markers [collagen type I (COL1A1), decorin (DCN), tenascin-C (TNC)], and biomechanical properties such as tendon stiffness and tensile strength. These results suggest that anti-inflammatory LF/Hep-PLGA NPs are effective at restoring tendons in Achilles tendinitis.

Association of estrogen receptor ß polymorphisms with posterior tibial tendon dysfunction.

Posterior tibial tendon (PTT) dysfunction is three times more common in females, and some patients may have a predisposition without a clinically evident cause, suggesting that individual characteristics play an important role in tendinopathy. The present study investigated the association of rs4986938 (+ 1730G > A; AluI RFLP) and rs1256049 (- 1082G > A; RsaI RFLP) single nucleotide polymorphisms (SNPs) of estrogen receptor-beta (ER-ß) gene with PTT dysfunction. A total of 400 participants were recruited. The PTT dysfunction group: these patients underwent surgery, with PTT tendinopathy confirmed by histopathology and magnetic resonance image (MRI). The control group was composed of participants with no clinical or MRI evidence of PTT dysfunction. Each group was composed of 100 postmenopausal women, 50 premenopausal women, and 50 men. Genomic DNA was extracted from saliva samples, and genotypes were obtained by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Concerning the ER-ß SNP rs4986938, there were significant differences in the frequencies of alleles between test and control groups of all the cases, only postmenopausal women and only men (p < 0.0001, p = 0.0016 and p = 0.0001). Considering the PTT dysfunction group and comparing postmenopausal women versus premenopausal women adding men, the analysis showed significant differences in the allelic distribution (p = 0.0450): the allele A in postmenopausal women is a risk factor. The ER-ß SNP rs1256049 did not show differences in the frequencies of alleles and genotypes between groups. The ER-ß SNP rs4986938, but not ER -ß SNPs rs1256049, may contribute to PTT insufficiency in the Brazilian population, with additional risk in postmenopausal women. Addition, in men the genetic factor could be more determinant.

Genetic factors in rotator cuff pathology: potential influence of col 5A1 polymorphism in outcomes of rotator cuff repair.

BACKGROUND: Investigations in genetics have provided valuable information about the correlation between gene variants and tendinopathy. Single Nucleotide Polymorphisms of COL5A1 gene are reported to be involved in Achilles tendinopathy, chronic degenerative tendon changes at the elbow, and other tendinopathies. The influence of Single Nucleotide Polymorphisms of COL5A1 was previously analyzed in rotator cuff disease with confounding results. Moreover, the rs12722 polymorphism in COL5A1 gene has been implicated in the aetiology of musculoskeletal soft tissue injuries in several association studies. This study aims to analyse the possible influence of rs12722 polymorphism in COL5A1 in the outcomes of rotator cuff repair. METHODS: Seventy-nine patients were included in the study. DNA was extracted from 1.2 ml of venous blood and genotyped for COL5A1 SNPs rs12722. Rotator cuff muscle strength and range of motion (ROM) in anterior elevation, external and internal rotation of the shoulder were evaluated. RESULTS: Patients presenting COL5A1 SNP rs12722 CC showed a ROM of passive external rotation statistically significantly higher compared to patients with CT genotype and TT genotype. CONCLUSIONS: COL5A1 SNP rs12722 may influence the functional outcomes of RCRs, even though further studies are required to confirm these preliminary results.

Relationship of common variants in MPP7, TIMP2 and CASP8 genes with the risk of chronic achilles tendinopathy.

Previous etiologic studies have indicated that both environmental and genetic factors play important roles in the occurrence and development of chronic Achilles tendinopathy (AT). A recent study documented the results of the largest genome-wide association study for chronic AT to date, indicating that MPP7, TIMP2 and CASP8 may be involved in the occurrence and development of chronic AT. In this study, we aimed to investigate whether MPP7, TIMP2 and CASP8 were associated with susceptibility to chronic AP in a Han Chinese population. A total of 3,680 study subjects comprised 1,288 chronic AT cases, and 2,392 healthy controls were recruited. Forty-four tag SNPs (7 from CASP8, 20 from MPP7, and 17 from TIMP2) were genotyped in the study. Genetic association analyses were performed at both single marker and haplotype levels. Functional consequences of significant SNPs were examined in the RegulomeDB and GTEx databases. Two SNPs, SNP rs1937810 (OR [95%CI] = 1.20 [1.09-1.32], χ2 = 13.50, P = 0.0002) in MPP7 and rs4789932 (OR [95%CI] = 1.24 [1.12-1.37], χ2 = 17.98, P = 2.23 × 10-5) in TIMP2, were significantly associated with chronic AT. Significant eQTL signals for SNP rs4789932 on TIMP2 were identified in human heart and artery tissues. Our results provide further supportive evidence for the association of the TIMP2 and MPP7 genes with chronic AT, which supports important roles for TIMP2 and MPP7 in the etiology of chronic AT, adding to the current understanding of the susceptibility of chronic AT.

Effects of tendon injury on uninjured regional tendons in the distal limb: An in-vivo study using an ovine tendinopathy model.

Following injury to a tendon little is known about potential for pathology to develop in other regional tendons from overloading or altered function. The aim of this study was to investigate the gene expression and histopathological changes that occur 1) within the deep digital flexor tendon (DDFT) after injury to the superficial digital flexor tendon (SDFT) and 2) within the flexor tendons (SDFT and DDFT) after injury to the extensor tendons. Merino wethers [Ovis aries] (n = 18) were divided into three equal groups and underwent either partial transection of the SDFT, complete transection of the extensor tendons or were left as non-operated controls. Tendons were harvested and sampled regionally for gene expression (real time PCR) and histologic analysis eight weeks after surgery. Transection of the SDFT resulted in increased expression of collagen III, versican, biglycan, lumican and MMP1 (P<0.026 for all genes) within the DDFT. There was no effect of transecting the extensor tendons on the expression of any gene tested in either the SDFT or the DDFT. The DDFT had elevated histopathology scores induced by transection of the SDFT, eight weeks previously. There were minimal histological differences in either the SDFT or DDFT after transection of the extensor tendons. Transection of the SDFT results in a mild, subclinical tendinopathy within the DDFT with potential implications on treatment and rehabilitation of SDFT injuries. Injury to the extensor tendons has minimal measured effect on the SDFT or DDFT.

S100A8 & S100A9: Alarmin mediated inflammation in tendinopathy.

Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease.

MicroRNA 148a-3p promotes Thrombospondin-4 expression and enhances angiogenesis during tendinopathy development by inhibiting Krüppel-like factor 6.

Tendinopathy is a common musculoskeletal disorder with characteristic hypervascularity. The mechanism of angiogenesis in tendinopathy remains unclear. The present study aimed to investigate the roles of miR-148a-3p in angiogenesis development of tendinopathy. In this study, we demonstrated that miR-148a-3p expression was increased in tendinopathy tissues and positively correlated with CD34 levels which is a specific marker for angiogenesis. We identified Krüppel-like factor 6 (KLF6) as a direct target gene of miR-148a-3p in tenocytes. Furthermore, reduced levels of KLF6 in tendinopathy tissues was showed using qRT-PCR and immunohistochemical analysis, compared with controls. A negative correlation between the levels of KLF6 mRNA and miR-148a-3p was observed. Then, we verified that miR-148a-3p could regulate Tsp-4 expression by targeting KLF6 in tenocyte and was positively correlated with Tsp-4 levels in tendinopathy tissues. In a coculture system of tenocytes with endothelial cells (ECs), we observed that transfection of Lv-miR-148a-3p markedly upregulated EC angiogenesis. In summary, our data establish a novel molecular mechanism by which miR-148a-3p upregulates Tsp-4 expression in tenocytes to promote EC angiogenesis by targeting KLF6, which could be helpful for the treatment of tendinopathy in the future.

Promoter methylation status of the TIMP2 and ADAMTS4 genes and patellar tendinopathy.

OBJECTIVES: Patellar tendinopathy (PT) is a debilitating and prevalent condition that tends to affect those who are physically active or engaged in jumping sports. Although tendinopathies are known to have a genetic basis, the role of DNA methylation as an epigenetic factor and risk determinant for human PT has never been described. We sought to determine whether differences existed between the methylation profiles of both the TIMP2 and ADAMTS4 gene promoter sequences in a cohort of males having undergone surgery for patellar tendinopathy compared to controls. DESIGN: Case-control epigenetic study using DNA from 10 males with PT and 10 males with healthy tendons. METHODS: We used PCR and targeted pyrosequencing to interrogate the methylation profiles of CpG sites upstream of both the TIMP2 (4 sites) and ADAMTS4 (6 sites) genes. We compared methylation differences between the two groups using t-tests. RESULTS: We report no significant (p>0.05) methylation differences within the TIMP2 gene promoter between the PT group and controls across the 4 CpG sites investigated. In contrast, we detected a significant (p=0.016) difference in the methylation status of 1 CpG site, approximately 3kb upstream of the ADAMTS4 gene between the PT group and controls. CONCLUSIONS: To our knowledge, this is the first study to investigate how DNA methylation impacts on the risk of human tendinopathy. Our data indicate that the methylation status of the ADAMTS4 gene is altered in patellar tendinopathy and we speculate on how this change might modify the patellar tendon extra-cellular matrix environment.

Mechanical loading modulates heterotopic ossification in calcific tendinopathy through the mTORC1 signaling pathway.

Excessive mechanical loading is a major factor affecting heterotopic ossification (HO), which is a major pathological alteration in calcific tendinopathy. However, physical therapies with mechanical loading as the functional element have exhibited promising results in the treatment of calcific tendinopathy. The dual effects that mechanical loading may have on the pathogenesis and rehabilitation of calcified tendinopathy remain unclear. The present study was designed to investigate the effects of mechanical loading on HO in calcific tendinopathy. In the present study, a tendon cell in vitro stretch model and an Achilles tenotomy rat model were used to simulate different elongation mechanical loading scenarios in order to investigate the effects of mechanical loading on HO of the tendon. In addition, rapamycin, a selective mammalian target of rapamycin complex­1 (mTORC1) signaling pathway inhibitor, was employed to determine whether mechanical loading modulates heterotopic ossification in calcific tendinopathy through the mTORC1 signaling pathway. The data indicate that mechanical loading modulated HO of the tendon through the mTORC1 signaling pathway, and that low elongation mechanical loading attenuated HO, while high elongation mechanical loading accelerated HO in vivo. This study may improve the understanding of the effect of physical therapies used to treat calcific tendinopathy, so as to guide clinical treatment more effectively. Furthermore, rapamycin may be a potential drug for the treatment of calcific tendinopathy.

Persistent stromal fibroblast activation is present in chronic tendinopathy.

BACKGROUND: Growing evidence supports a key role for inflammation in the onset and progression of tendinopathy. However, the effect of the inflammatory infiltrate on tendon cells is poorly understood. METHODS: We investigated stromal fibroblast activation signatures in tissues and cells from patients with tendinopathy. Diseased tendons were collected from well-phenotyped patient cohorts with supraspinatus tendinopathy before and after sub-acromial decompression treatment. Healthy tendons were collected from patients undergoing shoulder stabilisation or anterior cruciate ligament repair. Stromal fibroblast activation markers including podoplanin (PDPN), CD106 (VCAM-1) and CD248 were investigated by immunostaining, flow cytometry and RT-qPCR. RESULTS: PDPN, CD248 and CD106 were increased in diseased compared to healthy tendon tissues. This stromal fibroblast activation signature persisted in tendon biopsies in patients at 2-4 years post treatment. PDPN, CD248 and CD106 were increased in diseased compared to healthy tendon cells. IL-1ß treatment induced PDPN and CD106 but not CD248. IL-1ß treatment induced NF-κB target genes in healthy cells, which gradually declined following replacement with cytokine-free medium, whilst PDPN and CD106 remained above pre-stimulated levels. IL-1ß-treated diseased cells had more profound induction of PDPN and CD106 and sustained expression of IL6 and IL8 mRNA compared to IL-1ß-treated healthy cells. CONCLUSIONS: We conclude that stromal fibroblast activation markers are increased and persist in diseased compared to healthy tendon tissues and cells. Diseased tendon cells have distinct stromal fibroblast populations. IL-1ß treatment induced persistent stromal fibroblast activation which was more profound in diseased cells. Persistent stromal fibroblast activation may be implicated in the development of chronic inflammation and recurrent tendinopathy. Targeting this stromal fibroblast activation signature is a potential therapeutic strategy.

Polymorphisms within the COL5A1 gene and regulators of the extracellular matrix modify the risk of Achilles tendon pathology in a British case-control study.

Several genetic loci have been associated with risk of Achilles tendon pathology (ATP) within South African and Australian populations. The aim of this study was, therefore, to evaluate eight previously implicated genetic variants in an independent British population. A total of 130 asymptomatic controls (CON) and 112 participants clinically diagnosed with ATP comprising 87 individuals with chronic Achilles tendinopathy (TEN) and 25 with Achilles tendon ruptures (RUP) were included. All participants were genotyped for variants within the COL5A1, MIR608, IL-1ß, IL-6 and CASP8 genes. Primary findings implicated COL5A1 and CASP8. Three inferred allele combinations constructed from COL5A1 rs12722, rs3196378 and rs71746744 were identified as risk modifiers. The T-C-D combination was associated with increased risk of ATP (P = 0.023) and RUP (P < 0.001), the C-A-I combination was associated with increased risk of ATP (P = 0.011), TEN (P = 0.011) and RUP (P = 0.011) and the C-C-D combination was associated with decreased risk of ATP (P = 0.011) and RUP (P = 0.004). The CASP8 rs3834129 DD genotype was associated with decreased risk of TEN (P = 0.020, odds ratio: 0.45, 95% confidence interval: 0.22-0.90) and the CASP8 I-G (rs3834129-rs1045485) inferred allele combination was associated with increased risk of TEN (P = 0.031). This study further highlights the importance of polymorphisms within COL5A1 and CASP8 in the aetiology of ATP.