Degradation of elastic fiber and elevated elastase expression in long head of biceps tendinopathy.

Tendinopathy of the long head of the biceps (TLHB) involves various types of extracellular matrix degeneration, but previous studies have not evaluated elastic fibers. The purpose of this study was to investigate elastic fiber distribution in long head of the biceps (LHB). The TLHB tendons of 16 consecutive patients (eight men and eight women; average age of 55.75 years; age range of 40-71 years) were transected and harvested. Three cadaveric LHB tendons were used as the control group. The expression of collagen type I was decreased, but type III was increased in TLHB. Disruption of elastic fibers was particularly observed in grade II specimens where the level of elastase-positive staining was significantly higher than in grade I specimens. Elastic fibers were not observed in the grade III area, implying a higher expression of elastase than in the grade I area. Results of Western blotting showed that the expression of elastin was higher in the control group and the levels of elastin significantly decreased in grades II and III of TLHB. Levels of osteopontin and elastase were increased in primary culture of human tenocytes after experiencing elastic derived peptide treatment. These results suggested that elastase may be caused by the disruption of elastic fibers in the development of chronic tendinopathy and that elastic derived peptide may enhance elastase and osteopontin expression. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1919-1926, 2017.

Influence of repetitive mechanical loading on MMP2 activity in tendon fibroblasts.

Matrix metalloproteinase2 has been implicated in tendon pathology caused by repetitive movements. However, its activity in the early stages of the tendon's response to overuse, and its presence in the circulation as a possible indicator of tendon degradation, remain unknown. Human tendon cells were repetitively stretched for 5 days, and the rabbit Achilles tendon complex underwent repetitive motion 3× per week for 2 weeks. Quantitative polymer chain reaction analysis was performed to detect matrix metalloproteinase2/14 and tissue inhibitor of matrix metalloproteinase2 messenger ribonucleic acid of cells and rabbit tissue, and matrix metalloproteinase2 protein levels were determined with an enzyme linked immunoassay. Matrix metalloproteinase2 activity was examined using zymography of the conditioned media, tendon and serum. Immunohistochemistry was used to localize matrix metalloproteinase2 in tendon tissue, and the density of fibrillar collagen in tendons was examined using second harmonic generation microscopy. Tendon cells stretched with high strain or high frequency demonstrated increased matrix metalloproteinase2 messenger ribonucleic acid and protein levels. Matrix metalloproteinase2 activity was increased in the rabbit Achilles tendon tissue at weeks 1 and 2; however, serum activity was only increased at week 1. After 2 weeks of exercise, the collagen density was lower in specific regions of the exercised rabbit Achilles tendon complex. Matrix metalloproteinase2 expression in exercised rabbit Achilles tendons was detected surrounding tendon fibroblasts. Repetitive mechanical stimulation of tendon cells results in a small increase in matrix metalloproteinase2 levels, but it appears unlikely that serum matrix metalloproteinase2 will be a useful indicator of tendon overuse injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1991-2000, 2016.

Cathepsins in Rotator Cuff Tendinopathy: Identification in Human Chronic Tears and Temporal Induction in a Rat Model.

While overuse of the supraspinatus tendon is a leading factor in rotator cuff injury, the underlying biochemical changes have not been fully elucidated. In this study, torn human rotator cuff (supraspinatus) tendon tissue was analyzed for the presence of active cathepsin proteases with multiplex cysteine cathepsin zymography. In addition, an overuse injury to supraspinatus tendons was induced through downhill running in an established rat model. Histological analysis demonstrated that structural damage occurred by 8 weeks of overuse compared to control rats in the region of tendon insertion into bone. In both 4- and 8-week overuse groups, via zymography, there was approximately a 180% increase in cathepsin L activity at the insertion region compared to the controls, while no difference was found in the midsubstance area. Additionally, an over 400% increase in cathepsin K activity was observed for the insertion region of the 4-week overused tendons. More cathepsin K and L immunostaining was observed at the insertion region of the overuse groups compared to controls. These results provide important information on a yet unexplored mechanism for tendon degeneration that may operate alone or in conjunction with other proteases to contribute to chronic tendinopathy.

Early events of overused supraspinatus tendons involve matrix metalloproteinases and EMMPRIN/CD147 in the absence of inflammation.

BACKGROUND: The principal feature of tendon degeneration is structural change of the extracellular matrix (ECM) including collagens. In painful tendons, alterations of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been described; however, the initial molecular mechanism at the origin of these alterations is still poorly understood. A rat model of supraspinatus tendon overuse has been developed, which may be predictive of pathological tendon alterations. PURPOSE: To determine which MMPs are involved in early ECM remodeling during overuse and their relationship with the inflammatory context. STUDY DESIGN: Controlled laboratory study. METHODS: Analyses were performed on rat supraspinatus tendons at 2 and 4 weeks of overuse on a downhill treadmill. Transcript levels of MMPs and TIMPs were assessed by semiquantitative reverse transcription polymerase chain reaction. Western blotting and/or immunolabeling were used for MMP-2, MMP-3, MMP-13, and extracellular MMP inducer (EMMPRIN, also called cluster of differentiation [CD] 147) detection. In situ and/or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gelatin zymography was performed for MMP-2 and MMP-9. TIMP activity was revealed by reverse zymography. Inflammation was assessed by cytokine antibody array and/or immunolabeling. RESULTS: Compared with a control, overused supraspinatus tendons showed a significantly higher gelatinolytic activity at 2 weeks, which slightly decreased at 4 weeks. MMP-9 and MMP-13 were undetectable; MMP-3 was downregulated in overused tendons. Only MMP-2, particularly its active form, and the MMP-2 activator MMP-14 were upregulated at 2 weeks of overuse when an increase in TIMP-2 transcripts was observed. MMP-2 upregulation occurred in the absence of inflammation but was associated with an increase of EMMPRIN/CD147. CONCLUSION: EMMPRIN/CD147-regulated MMP-2 and MMP-14, associated with low MMP-3, appear as the main characteristics of ECM remodeling in early overused tendons. Whether alterations in the pattern of these MMPs are an adaptive response or a repair response that may degenerate into tendinosis, is still uncertain. Moreover, there seems to be no indication for an inflammatory response to overuse, suggesting that the increased metalloproteinase activity is rather a response to a mechanical stress than an inflammatory one. CLINICAL RELEVANCE: Any strategy aimed at preventing full-thickness tears resulting from initial tendon matrix alterations should consider these changes in MMP-3, MMP-2, and MMP-14, or further upstream, EMMPRIN.

Active hyaluronidase 2 expression in the granulation tissue formed in the healing process of equine superficial digital flexor tendonitis.

Upregulation of hyaluronidase 2 (HYAL2), one of somatic hyaluronidase (HAase), was demonstrated in granulation tissue during the healing of equine superficial digital flexor tendon injuries. The activity of HAase was assessed by hyaluronan (HA)-containing gel zymography and in situ zymography using frozen sections obtained from normal and injured tendon tissues. Elevated HAase activity was identified in the extract from the tendinopathic tissues, with lower levels of the activity in normal tendons. In situ zymography using fluorescently-labeled HA demonstrated HAase activity in the granulation tissue formed in the injured region. In addition, in situ hybridization analysis indicated that fibroblastic cells in the granulation tissue of the injured tendon actively expressed HYAL2 but not HYAL1. Quantitative RT-PCR further confirmed a higher level of amplicons corresponding to HYAL2 in tendonitis-derived samples. These results suggest that elevated HYAL2 activity in the granulation tissue could participate in controlling the healing process in equine tendonitis.

Sirt-1 is required for the inhibition of apoptosis and inflammatory responses in human tenocytes.

Tendon overuse injuries and tendinitis are accompanied by catabolic processes and apoptosis of tenocytes. However, the precise molecular mechanisms of the destructive processes in tendon are not fully understood. Sirt-1, a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, has been linked to transcriptional silencing and appears to play a key role in inflammation. The purpose of this study was to examine whether down-regulation of Sirt-1 using antisense oligonucleotides (ASO) affects inflammatory and apoptotic signaling in tenocytes. Transient transfection of tenocytes with ASO against Sirt-1 induced expression of Bax and other proteins involved in apoptosis (cleaved caspase-3 and poly(ADP-ribose)polymerase), acetylation of tumor suppressor p53, and mitochondrial degradation. Interestingly, Sirt-1 was found to interact directly with p53. In contrast, Sirt-1 activator resveratrol inhibited interleukin-1ß (IL-1ß)- and nicotinamide-induced NF-κB activation and p65 acetylation and suppressed the activation of IκB-α kinase. Resveratrol also reversed the IL-1ß- or nicotinamide-induced up-regulation of various gene products that mediate inflammation (cyclooxygenase-2) and matrix degradation (matrix metalloproteinase-9) that are known to be regulated by NF-κB. Knockdown of Sirt-1 by using ASO abolished the inhibitory effects of resveratrol on inflammatory and apoptotic signaling including Akt activation and SCAX suppression. Down-regulation of histone deacetylase Sirt-1 by mRNA interference abrogated the effect of resveratrol on NF-κB suppression, thus highlighting the crucial homeostatic role of this enzyme. Overall, these results suggest for the first time that Sirt-1 can regulate p53 and NF-κB signaling via deacetylation, demonstrating a novel role for resveratrol in the treatment of tendinitis/tendinopathy.

Metalloproteases and rotator cuff disease.

The molecular changes occurring in rotator cuff tears are still unknown, but much attention has been paid to better understand the role of matrix metalloproteinases (MMP) in the development of tendinopathy. These are potent enzymes that, once activated, can completely degrade all components of the connective tissue, modify the extracellular matrix (ECM), and mediatethe development of painful tendinopathy and tendon rupture. To control the local activity of activated proteinases, the same cells produce tissue inhibitors of metalloproteinases (TIMP) that bind to the enzymes and prevent degradation. The balance between the activities of MMPs and TIMPs regulates tendon remodeling, whereas an imbalance produces a collagen dis-regulation and disturbances intendons. ADAMs (a disintegrin and metalloproteinase) are cell membrane-linked enzymes with proteolytic and cell signaling functions. ADAMTSs (ADAM with thrombospondin motifs) are secreted into the circulation, and constitute a heterogenous family of proteases with both anabolic and catabolic functions. Biologic modulation of endogenous MMP activity to basal levels may reduce pathologic tissue degradation and favorably influence healing after rotator cuff repair. Further studies are needed to better define the mechanism of action, and whether these new strategies are safe and effective in larger models.

Prostaglandin E(2), collagenase, and cell death responses depend on cyclical load magnitude in an explant model of tendinopathy.

Tendinopathy is a significant clinical problem that can result from repetitive activity. While the precise etiology of this condition remains unclear, the cellular response to cyclical loading is believed to have a contributory role to the pathology of tendinopathy. This study examined the short-term biochemical response of avian flexor digitorum profundus tendon to repetitive cyclic loadings of varying magnitude. An in vitro tendon explant model was utilized to apply four levels of haversine tensile stress (peak stress of 0, 3, 12, and 18 MPa) at 1.0 Hz, 8 hr/day for 3 days. The 12 and 18 MPa levels were known to cause significant mechanical damage based on previous work. Tissue media was recovered and analyzed for prostaglandin E(2) (PGE(2)), lactate dehydrogenase (LDH, measure of cell death), and collagenase levels. Tissue samples were recovered and analyzed for cell viability, total collagen, and sulfated glycosaminoglycan content. Collagenase, LDH, and PGE(2) levels were found to be influenced by loading magnitude (p < 0.05) with higher levels being present at higher load magnitudes. Varying cyclical load magnitude caused minimal compositional changes as collagen content and glycosaminoglycan did not change. These results indicate that elevated cyclical mechanical loading of tendon quickly results in altered biochemical tissue responses indicative of tissue injury. More sustained cyclical loading over time may be required for these initial responses to induce more dramatic tissue changes as observed in clinical tendinopathy.

Cell phenotypic variation in normal and damaged tendons.

Injuries to tendons are common in both human athletes as well as in animals, such as the horse, which are used for competitive purposes. Furthermore, such injuries are also increasing in prevalence in the ageing, sedentary population. Tendon diseases often respond poorly to treatment and require lengthy periods of rehabilitation. The tendon has a unique extracellular matrix, which has developed to withstand the mechanical demands of such tensile-load bearing structures. Following injury, any repair process is inadequate and results in tissue that is distinct from original tendon tissue. There is growing evidence for the key role of the tendon cell (tenocyte) in both the normal physiological homeostasis and regulation of the tendon matrix and the pathological derangements that occur in disease. In particular, the tenocyte is considered to have a major role in effecting the subclinical matrix degeneration that is thought to occur prior to clinical disease, as well as in the severe degradative events that occur in the tendon at the onset of clinical disease. Furthermore, the tenocyte is likely to have a central role in the production of the biologically inadequate fibrocartilaginous repair tissue that develops subsequent to tendinopathy. Understanding the biology of the tenocyte is central to the development of appropriate interventions and drug therapies that will either prevent the onset of disease, or lead to more rapid and appropriate repair of injured tendon. Central to this is a full understanding of the proteolytic response in the tendon in disease by such enzymes as metalloproteinases, as well as the control of the inappropriate fibrocartilaginous differentiation. Finally, it is important that we understand the role of both intrinsic and extrinsic cellular elements in the repair process in the tendon subsequent to injury.

In situ hybridization studies confirming recent findings of the existence of a local nonneuronal catecholamine production in human patellar tendinosis.

We have in recent studies presented unexpected immunohistochemical evidence favoring the existence of a local production of catecholamines, and an occurrence of adrenergic receptors on the tendon cells (tenocytes), in the human patellar tendon. This was particularly noticed for tendons from patients suffering from tendinosis (chronic tendon pain), which has led us to propose an involvement of this autocrine/paracrine system in the development of tendinosis, especially since catecholamines have been reported to be modulators of tissue remodeling and pain processes. However, the findings concerning catecholamine production have so far only been noted at the level of protein detection, and for this reason, the aim of the present study was to confirm the previous immunohistochemical results by using in situ hybridization (ISH) technique. A ssDNA probe detecting human mRNA for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) was applied. The ISH results revealed that there were clear reactions indicating the existence of mRNA for TH in tenocytes of tendinosis specimens. It was generally noted that disfigured tenocytes were the ones with the most distinct reactions, while normally looking tenocytes hardly displayed any reactions at all. In conclusion, this study presents the first evidence at the mRNA level of the existence of a local nonneuronal production of catecholamines in human patellar tendon tissue. The findings add to recent observations of the occurrence of a local production in tendons of signal substances traditionally related to neurons.

Overexpression of nitric oxide synthases in tendon overuse.

Tendon disorders with a chronic nature, including the rotator cuff, are extremely common, and represent a major clinical problem. Mechanical overload has been proposed as an important etiologic factor in tendinopathies. Nitric oxide (NO), a free radical produced by nitric oxide synthases (NOSs), is a potent regulator and stimulator of biological processes including tendon degeneration and healing. It is also involved in response to mechanical stimuli in different tissues. In an animal model of acutely injured tendon healing temporal and differential expression of NOS isoforms has been demonstrated, suggesting that different patterns of NOSs expression may have different biological functions. Therefore, we hypothesized that tendon overuse may result in a differential upregulation of NOSs, particularly iNOS. An animal model of supraspinatus tendon overuse was utilized, which consisted of treadmill running. A group of animals of the same strain and age subjected to normal cage activity were used as controls. Following a 4-week exercise protocol supraspinatus tendons were harvested, RNA was extracted, and subjected to competitive reverse transcription and polymerase chain reaction (RT-PCR) to determine the expression levels of inducible-, endothelial-, and neuronal-NOS isoforms (i-, e-, and nNOS). The mRNA expression of all three NOS isoforms increased in the supraspinatus tendons as a result of overuse exercise. iNOS and eNOS mRNA expression increased fourfold (p < 0.01), and there was an increase, but statistically not significant, in nNOS mRNA expression in the overused tendons when compared with the controls. This study is the first to show that NOS isoforms are upregulated in rotator cuff tendon as a result of chronic overuse, and suggests the involvement of nitric oxide in the response of tendon tissue to increased mechanical stress.

Cell and matrix changes associated with pathological calcification of the human rotator cuff tendons.

The causative mechanism of tendon calcification ('calcifying tendinitis') is unknown. In this report, pathological human tendon samples were examined to give morphological and ultrastructural detail of the calcified regions and these findings were compared with those from normal tendon. Selected specimens were cryosectioned to enable histochemical and immunohistochemical comparison of the occurrence and distribution of specific matrix molecules in diseased and normal tendon tissues. The lack of collagen type II and alkaline phosphatase in the pathological regions suggests that the calcification process is not mediated through an endochondral transition. In contrast, the pathological areas were characterised by widespread labelling for chondroitin-4-sulphate/dermatan sulphate and intense pericellular localisation of chondroitin-6-sulphate.

Chronic Achilles paratenonitis in athletes: a histological and histochemical study.

Pathological alterations of chronic Achilles paratenonitis were studied histologically and histochemically in tissue samples obtained operatively from 16 athletes with this complaint and from 3 control patients. The activities of 11 different enzymes--lactate, succinate, malate, glucose-6-phosphate and glutamate dehydrogenases, lipoamide dehydrogenase and glutathione reductase (NADH2- and NADPH2-diaphorases), acid and alkaline phosphatases, phosphorylase and leucylaminopeptidase--were studied. Pathological findings were located diffusely around the tendon. A slight inflammatory cell reaction was found in all cases. The fatty areolar tissue was clearly thickened and edematous, and showed fibrinous exudations, widespread fat necrosis, considerable connective tissue proliferation and adhesion formation. The blood vessels showed profound degenerative and necrotizing changes. The thin membranes of the paratenon were clearly hypertrophied. Increased enzyme activities were mainly found in the fibroblasts, inflammatory cells and vascular walls. A moderate activity of lysosomal enzymes, an increased activity of enzymes of electron transport, anaerobic glycolysis, pentose phosphate shunt and decreased activity of those of aerobic energy metabolism were found. Simultaneously an increased amount of both neutral and acid mucopolysaccharides and a locally increased amount of elastic fibres were found in the inflamed paratenon. These results indicate that marked metabolic changes occur in paratenonitis, i.e. an increased catabolism and decreased oxygenation of the inflamed areas. The morphological alterations suggest that the gliding function of the paratenon may be impaired.