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J Appl Physiol 101: 1702-1709, 2006. First published August 3, 2006; doi:10.1152/japplphysiol.00386.2006
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Microarray analysis of the tendinopathic rat supraspinatus tendon: glutamate signaling and its potential role in tendon degeneration

T. J. Molloy, M. W. Kemp, Y. Wang, and G. A. C. Murrell

Orthopaedic Research Institute, St. George Hospital Campus, University of New South Wales, Sydney, Australia

Submitted 31 March 2006 ; accepted in final form 22 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Degenerative tendon injury or "tendinopathy" is one of the most common disorders of the musculoskeletal system. We used a rat model (Soslowsky LJ, Thomopoulos S, Tun S, Flanagan CL, Keefer CC, Mastaw J, and Carpenter JE. J Shoulder Elbow Surg 9: 79–84, 2000) to identify novel gene expression in the exercised-induced degenerated supraspinatus tendon by microarray and real-time PCR analyses. We identified several novel groups of differentially expressed genes, including those involved in apoptosis and related stress responses, and also genes that appear to be involved in glutamate signaling in tendon tissue, similar to recent findings by us in a microarray study of healing in the transected Achilles tendon of the rat (24). Until recently this kind of cellular communication was thought only to exist in cells of the central nervous system (CNS), where it is vital for CNS function. We further show that glutamate appears to induce a proapoptotic response in cultured tendon cells, similar to the "excitotoxic" response of cells in the CNS that become overstimulated. This may prove to be at least a partial cause of degeneration in overused tendon tissue and allow the development of treatments or "prehibilitation" regimens for tendinopathy based on currently used non-toxic glutamate antagonists.

tendinopathy; excitotoxicity; gene expression; apoptosis

TENDINOPATHY IS THE COLLECTIVE term used to describe degenerative damage to tendon tissue and is characterized by inflammation, plastic deformation, and/or microtearring. It includes the disorders tendinosis and tendinitis and often results in pain and tenderness, as well as a loss of biomechanical strength and range of movement in the anatomical structures involved. The supraspinatus tendon of the rotator cuff is one of the most commonly affected tendons of the musculoskeletal system and becomes degenerated most often as a result of repetitive stresses and overloading during sports or occupational activities (30). A variety of treatments is currently used or has been trialled, including steroid injections (35), ultrasound (9), nonsteroidal anti-inflammatory drugs (NSAIDs; 23), hyperbaric oxygen therapy (14), and extracorporeal shock wave therapy (37); however, complete functional recovery of the damaged tissue is rarely seen. Although histological observations such as collagen fibril disorientation (18, 36), vascular changes (17), and hypercellularity of the affected tissue (18) have been noted, the molecular and genetic etiology of this disorder remains poorly understood. A better understanding of the interactions that occur at a cellular and molecular level during tendon degeneration may allow the development of more targeted therapeutics and result in a greater number of positive clinical outcomes.

The relatively recent development of an appropriate animal model (31) has aided study into supraspinatus tendinopathy. Rats exercised daily on a treadmill for as little as 4 wk demonstrate degenerative damage to the supraspinatus tendon comparable to that seen in human tedinopathic patients. Gross morphological characteristics, collagen organization, cross-sectional area, and biomechanical properties all become significantly altered compared with animals at a normal level of activity, closely resembling the degenerated tendon in affected humans (31). The current study used this model together with microarray analyses in an attempt to identify genes and genetic pathways that may contribute to the progression of degeneration in overused tendons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal model and sample collection.   Twenty-four male, 30-wk-old Sprague-Dawley rats weighing 342–388 g each were randomly divided equally into exercise and control groups. The exercise group underwent a daily treadmill running regimen to model tendon overuse resulting in degeneration as previously described (31). Rats initially underwent daily training increasing in duration for 2 wk to accustom them to the exercise and surroundings. Subsequent to this, rats were subjected to exercise that consisted of running on a 10° decline at 17 m/min for 1 h/day, 5 days/wk. This regimen equates to ~7,500 strides/day. After 4 wk of running, rats were killed by CO2 inhalation and both supraspinatus tendons were collected. The control group consisted of 12 nonexercised rats.

RNA isolation.   Total RNA was isolated from tendon tissue using Trizol Reagent (Invitrogen Life Technologies, Victoria, Australia) as per the manufacturer's instructions. RNA yield and integrity was evaluated by spectrophotometry and agarose gel electrophoresis, respectively.

Microarray hybridization.   Microarrays consisting of 5,760 rat oligonucleotide features in duplicate were provided by the Ramaciotti Centre of the University of New South Wales (Sydney, Australia). Fiteen to twenty micrograms of total RNA extracted from both supraspinatus tendons of two exercised animals (4 tendons) was pooled and used to synthesize experimental cDNA for each microarray. Total RNA extracted from four supraspinatus tendons from nonexercised animals were similarly pooled and used as controls for each microarray. Cy3 or Cy5 fluorescent dyes (Amersham Pharmacia Biotech, North Ryde, New South Wales, Australia) were incorporated using an indirect labeling method. Control and experimental labeled cDNAs were then cohybridized overnight to each of three microarrays. One of the three microarrays was labeled in reverse to the other two (for example Cy3 used to label control instead of experimental cDNA—a "dye swap" control measure) in an attempt to lower dye-bias effects. Microarrays were scanned using an Axon GenePix 4000B microarray scanner (Molecular Devices, Union City, CA), and the resulting image was analyzed using GenePix Pro software version 3.0.

Microarray data analysis.   All genes were represented by at least two independent targets on each microarray (i.e., at least 6 independent targets across the 3 microarrays), and the signal from each target was used to calculate an average signal for each gene. A gene was considered upregulated or downregulated if it had at least a twofold difference from controls, an average rank in the top or bottom 10% of all genes, and was significant according to the Wilcoxin paired-sample test at the 0.05 level. The mining of microarray data was facilitated by the Combina.tions software developed by Chris Molloy. This software is available online and free to use at http://www.combina.tions.net.

RT-PCR.   Total RNA extracted was used as a template to generate cDNA using the SuperScript III reverse transcriptase system (Invitrogen Life Technologies) as per manufacturer's instructions.

Quantitative PCR.   Quantitative PCR reactions were performed using the Rotorgene 3000 (Corbett Research, Mt. Waverly, Australia) using the dsDNA binding fluorescent dye, SYBR Green I. PCR reactions were performed in 0.1 ml thin-walled PCR tubes (Corbett Research). The sequence of primers (5' -> 3') and their annealing temperatures used to amplify cDNA regions are shown in Table 1.


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  		Table 1. Sequences and annealing temperatures of primers used to amplify regions of genes for the quantitative real-time PCR analyses

 
All amplifications were performed at a primer concentration of 200 nM and Mg2+ concentration of 2 mM. Platinum QPCR SYBR Green Mix [SYBR Green I fluorescent dye, 60 U/ml Platinum Taq DNA polymerase, 40 mM Tris·HCl (pH 8.4), 100 mM KCl, 6 mM MgCl2, 400 µM dGTP, 400 µM dATP, 400 µM dCTP, 400 µM dUTP, 40 U/ml UDG, stabilizers; Invitrogen Life Technologies] was used together with primers and 50 pg–50 ng of template. The thermal cycling program consisted of an initial denaturation step of 95°C for 1:15 s, followed by 50 cycles of 95°C for 45 s, a 30-s annealing step at varying temperatures, and a 30-s extension step at 73°C. Fluorescent readings were taken during the extension step of each cycle. Melting curve analysis was also performed to ensure the amplification of a single PCR product. Reactions with no RT enzyme and no template were included as negative controls. Each sample for each gene consisted of six to eight replicates.

All samples were calculated relative to the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, which was also amplified for each sample. This was selected as an acceptable endogenous reference "housekeeping" gene as its expression was found not to be significantly different (P = 0.92) in the 120 features representing GAPDH found on the microarrays (40 features per array).

Sample collection and cell culture conditions.   Samples of normal supraspinatus tendon were collected from rats and washed with sterile Dulbecco's PBS, cut into small pieces, and digested in collagenase solution [0.125 mg/ml type IA clostridium histolyticum collagenase, 2% (vol/vol) 1 M HEPES, 2% (vol/vol) antibiotic-antimycotic solution, in Hanks' balanced salt solution], pelleted, and then resuspended in cell-culture medium [DMEM with 10% (vol/vol) FCS and 1% (vol/vol) antibiotic-antimycotic solution] before being plated into flasks. Culture condition was kept constant at 37°C in a 5% CO2 humidified atmosphere. Media were changed after 48 h, then twice weekly. After the cells had become confluent, they were trypsinized and passaged into new flasks. Second- or third-passage cells were used for all RT-PCR and cell culture work.

Cell treatments.   For glutamate stimulation, cells were exposed to 500 µM glutamate (6) for 24 h. For glutamate receptor inhibition studies, the antagonist MK801 was added to cells at a concentration of 1 µM 1 h before glutamate was added. Hydrogen peroxide (500 µM) was used as a positive control in the caspase-3 activity assays; as we have previously shown, H2O2 can trigger apoptosis in cultured tendon cells (38). DNase-treated cells were used as a positive control for the TdT-mediated dUTP nick-end labeling (TUNEL) assay as per manufacturer's instructions. The pan-caspase inhibitor z-VAD-fmk was added 24 h before some treatments at a 50 µM concentration as per manufacturer's instructions.

TUNEL assay.   Apoptotic cells were identified by the use of the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI), which detects nuclear DNA fragmentation, an important indicator of apoptosis, as per manufacturer's instructions. Primary rat supraspinatus tendon fibroblasts were cultured and passaged onto poly-L-lysine-coated microscope slides. After being allowed to settle for 24 h, cells were either stimulated with glutamate or glutamate + z-VAD-fmk. Untreated cells were used as controls and H2O2-treated cells used as a positive control. Negative controls lacked the addition of the terminal deoxynucleotidyl transferase enzyme, which allows the labeling of fragmented DNA in the apoptotic cells.

Caspase-3 activity assay.   Caspase-dependant apoptosis was demonstrated by the measurement of caspase-3 activity using the Colorimetric CaspACE Assay System (Promega) as per manufacturer's instructions. The colorimetric substrate Ac-DEVD-pNA releases pNA, which produces a yellow color on cleavage by caspase-3. This can be detected by spectrophotometry and is proportional to the amount of caspase-3 activity present in the sample. Caspase-3 activity was measured in samples after stimulation with glutamate. The potent, irreversible, and cell-permeable pan-caspase inhibitor z-VAD-fmk was also simultaneously added to some samples to inhibit caspase-3 activity. Untreated cells were used as controls and H2O2-treated cells were used as a positive control.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Microarray analyses.   Of the 5,760 independent targets on each of the microarrays, the majority had less than a twofold change in expression in the experimental samples vs. the controls and thus were considered not differentially expressed. Overall, 91 genes were found to be significantly upregulated, and 37 significantly downregulated. Table 2 lists the genes likely to be of most importance to the tendinopathic state that were significantly differentially expressed, with their accession numbers and level of up- and downregulation. Of note was the differential expression of inflammation-related genes, representing 12% (11 genes) of the significantly upregulated genes and 19% (7 genes) of significantly downregulated genes. Also differentially expressed were cellular communication genes, including members of the glutamate signaling machinery, representing 13% (12 genes) of the genes found to be significantly upregulated and 8% (3 genes) of significantly downregulated genes. Several growth, differentiation, and developmental genes were also differentially regulated in the degenerated tendons, representing 14% (13 genes) of upregulated and 11% (10 genes) of downregulated genes. Complete microarray data can be found in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database in MIAME-compliant form under the accession number GSE4575 or, alternatively, at the web address http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE4575.


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  		Table 2. Genes that were significantly differentially expressed in degenerated rat supraspinatus tendon compared with controls that were likely to be of most importance to the tendinopathic state, with their accession numbers and level of up- or downregulation

 
Quantitative real-time PCR analyses.   Of the genes identified by microarray analysis to be significantly differently expressed in degenerated tendon vs. controls, two groups of interest were chosen and their expression confirmed by SYBR Green real-time PCR analyses (Fig. 1, A and B), glutamate signaling genes, and apoptosis- and stress-related genes. Of the glutamate signaling genes measured (Fig. 1A), the metabotropic glutamate receptor (mGluR) genes mGluR5 and mGluR6 showed a significant upregulation in degenerated tendon of 2.4- and 3.2-fold, respectively. Likewise, the N-methyl-D-aspartate receptor (NMDAR) homolog NMDA receptor-like 1 (NMDARL1) showed a significant 2.4-fold upregulation. Glutamate receptor interacting proteins (GRIP)-1 and -2 both showed a significant downregulation of 1.8- and 3.6-fold, respectively, in degenerated tendon. Of the apoptosis and stress-related genes found to be differentially regulated in degenerated tendon by microarray analysis, poly(ADP-ribose) polymerase (PARP) showed a downregulation of 1.6-fold by quantitative real-time PCR measurement, and flice inhibitory protein (FLIP), heat shock protein 27 (HSP27), and testis heat shock-related protein 70 (HST70) all showed significant upregulation of 4.1-fold, 7.1-fold, and almost 33-fold, respectively (Fig. 1B).


Figure 1
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  		Fig. 1. Two groups of genes of particular interest were confirmed by quantitative real-time PCR analyses: glutamate signaling genes (A) and apoptosis and stress-related genes (B). All were confirmed to be significantly differentially regulated in degenerated tendons compared with controls (*P < 0.05, **P < 0.01). Values are expressed as a percentage of GAPDH expression ± SD and are an average of 8 animals for the experimental (degenerated) group and 4 animals for the control group, each measured in triplicate during real-time PCR.

 
These apoptosis and stress genes were also measured in an in vitro culture of rat supraspinatus tendon fibroblasts in response to glutamate, and glutamate after pretreatment with the glutamate receptor antagonist MK801, with untreated and MK801-only treated cells as controls (Fig. 2). Neither the expression of HSP27 or HST70 was significantly altered by glutamate treatment compared with untreated controls, whereas FLIP showed a small (28%) but significant increase in expression, and PARP demonstrated a small but significant (13%) decrease in expression. MK801-only treatment showed no significant effect on the expression of the genes assayed.


Figure 2
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  		Fig. 2. Expression of the apoptosis and stress-response genes identified from the microarray analyses was measured in cultured rat supraspinatus tendon fibroblasts in response to glutamate treatment by quantitative real-time PCR (*P < 0.05). Open bars, controls; solid bars, glutamate treatments; dark gray bars, glutamate + MK801 treatments; light gray bars, MK801-only treatments. Values are expressed as a percentage of GAPDH expression ± SD and are an average of 4 independent experiments, each measured in triplicate during real-time PCR.

 
RT-PCR.   To demonstrate that the tendon fibroblasts themselves were expressing the glutamate signaling genes identified by microarray and were not found to be upregulated simply due to the inadvertent collection of nerve or other tissue with the tendons, RT-PCR for each gene was performed on a cDNA template generated from total RNA extracted from a pure culture of rat supraspinatus tendon. All genes were in fact found to be expressed by tendon fibroblasts (see Fig. 3).


Figure 3
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  		Fig. 3. To ensure that the expression of the glutamate signaling genes was a result of their presence in the tendon fibroblasts themselves and not seen as a result of inadvertent collection of nerve tissue within the degenerated tendon area, RT-PCR was performed for each gene using RNA extracted from a pure culture of rat supraspinatus tendon fibroblasts. All 5 genes were detected in tendon fibroblasts: A, mGluR5; B, mGluR6; C, NMDARL1; D, GRIP1; E, GRIP2.

 
TUNEL assay.   TUNEL staining revealed clusters of dark brown nuclear-stained cells in both glutamate-treated and the positive control H2O2- and DNase-treated cells. Positively stained cells markedly decreased in number in cultures pretreated with z-VAD-fmk in both cases. No staining was present in either negative controls or non-treated cells (Fig. 4).


Figure 4
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  		Fig. 4. Cultured supraspinatus tendon fibroblasts underwent a TdT-mediated dUTP nick-end labeling (TUNEL) assay to detect nuclear DNA degradation, a feature of apoptotic cells. Cells with brown-stained nuclei are positive for DNA degradation. Glutamate-treated fibroblasts were positively stained (B), similar to the H2O2 (D)- and DNase-treated (F) positive control cells. The pan-caspase inhibitor z-VAD-fmk decreased apoptosis in glutamate (C)- and H2O2-treated (E) cells to control levels (A). Negative control cells (G), which were treated with the TUNEL reaction mix lacking the terminal deoxynucleotidyl transferase enzyme, are not stained.

 
Caspase-3 activity assay.   Caspase-3 activity assays provided further evidence of apoptotic processes within treated cells (Fig. 5). Glutamate-treated tendon cells showed a significant increase in caspase-3 activity compared with controls, which was returned to levels near that of controls when treated with z-VAD-fmk. H2O2 also elicited an increase in caspase-3 activity, which was again decreased by treatment with z-VAD-fmk. Levels did, however, remain slightly, but significantly, higher than untreated controls.


Figure 5
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  		Fig. 5. Caspase-3 activity was also measured in glutamate-treated cells. Glutamate- and H2O2 (positive control)-treated cells both showed a significant (*P < 0.05) increase in caspase-3 activity compared with non-treated controls and cells concurrently treated with the pan-caspase inhibitor z-VAD-fmk. Data expressed as the average absorbance of 3 independent samples, each measured in duplicate, divided by the total amount of protein in each sample ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Gene expression in tendinopathic tendon.   We used a rat model of supraspinatus tendinopathy to identify novel gene expression in degenerated tendon by microarray and real-time PCR analyses. A range of gene expression was observed, with several groups of genes potentially involved in important aspects of tendinopathy. Significant increases in the expression of a variety of immune response genes were observed (17 of the 93 genes found to be upregulated and 9 of the 37 genes found to be downregulated in the degenerated tendon were related to the inflammatory response), particularly immunoglobulins and inflammatory cell receptors. This finding is consistent with other studies, which suggest that mechanical loading of tendon fibroblasts induces the production of stimulators of inflammatory mediators such as leukotrienes (20), 5-lipoxygenase (20), and prostaglandin E2 (3, 4). Inflammation is often (although not always) a feature of tendinopathy and leads to painful swelling of the affected area and may also contribute to further tissue degradation.

Genes encoding growth factors and proproliferative molecules are also significantly upregulated in the current study, including fibroblast growth factors (FGFs) and their receptors (FGFRs), interleukins (ILs), and transforming growth factor-beta (TGF-beta)-related proteins. These molecules are likely coordinating growth and proliferation of both endogenous fibroblasts and inflammatory cells in the affected area. Some of these molecules, such as FGF-18, FGFR1, IL-11, and TGF-beta binding protein 1, may also be involved in the regulation of apoptosis (10, 12, 19, 33), which, as discussed below, is likely to be an important aspect of the pathophysiology of tendinopathic tendons.

Glutamate signaling machinery in tendinopathic tendon.   Several genes are upregulated whose proteins constitute various members of well-characterized glutamate signaling machinery, which until relatively recently was only thought to exist in the central nervous system (CNS). We have recently identified and measured the expression of some of these genes in an acute injury healing model (24; in which the Achilles tendon of the rat was fully transected and allowed to heal), so it is of interest to see them also upregulated in a tendinopathy model. Their recent discovery and characterization in bone (21, 26) were the first time that these types of signaling mechanisms were shown to exist outside the CNS. It has since been shown that osteoblasts express a range of glutamate signaling proteins that appear to function in bone in an analogous fashion to synaptic transmission between neurons (5, 29, 32). Here we have used real-time PCR analyses to show that metabotropic glutamate receptor-5 (mGluR5) and -6 (mGluR6) and the N-methyl-D-aspartate acid (NMDA) receptor-like 1 (NMDARL1) gene, thought to be a member of the ionotropic glutamate receptor family, are significantly upregulated in degenerated tendon compared with non-degenerated controls (Fig. 1A). The accessory proteins glutamate receptor interacting protein-1 (GRIP1) and -2 (GRIP2), which are thought to bind ionotropic receptors and help target them to the cell surface (8), were both significantly downregulated (Fig. 1A). To ensure that these genes were in fact expressed by tendon cells and did not appear upregulated as a result of inadvertent collection of neuronal or other tissue during tendon excision, a pure sample of rat supraspinatus fibroblasts was cultured, and all genes were detected by RT-PCR in RNA extracted from them (Fig. 3). Previous work in our laboratory has also used immunohistochemistry to demonstrate protein localization to fibroblast cells in the tendon (24). Others have shown that in osteoblasts, these genes are expressed together with all the minimum requirements for core exocytotic complex formation, and that these colocalize as in functional glutamate systems in the CNS (5). Previous studies by others have also shown that numerous accessory proteins are expressed with these genes, and electrophysiological studies have demonstrated that the glutamate receptors are fully functional (11). We previously showed that the accessory proteins rSec8, homer-1b, homer3, and syn2b are expressed in rat Achilles tendon and are upregulated following acute injury (24). It is, therefore, likely that the genes shown to be differentially regulated here are also functional in tendon tissue.

This is the first time that these sorts of mechanisms have been suggested to play some role in the tendinopathic tendon, although the exact significance of these types of signaling mechanisms remains unknown. In bone they are upregulated in response to mechanical loading (29; which may be significant as repetitive loading of tendon tissue directly results in tendinopathy) and have also been postulated to modulate osteoblastic differentiation during cellular maturation (13). It has further been hypothesized that this CNS-like signaling may constitute a cellular "memory" to allow long-term changes to take place in the tissue in response to short-term mechanical stresses (for example, adaptive osteogenesis in bone in response to mechanical loading, which allows the bone to be better adapted for subsequent loading events; 32). Each of these suggestions would seem to agree with their observed upregulation in acute and overuse tendon injury models.

Excitotoxicity in tendinopathic tendon?   Overactive glutamate signaling in the neuronal cells of the CNS ("excitotoxicity") is extremely deleterious for cells (25). Under normal circumstances, the effects of glutamate toxicity are kept in check by its constant cellular sequestration via a high-affinity uptake system that removes glutamate from the extracellular space (7). Once the extracellular concentration reaches a certain threshold, however, affected cells may either undergo rapid cell swelling followed by lysis [within minutes of exposure (27)] or a more slowly evolving cellular degeneration. There is increasing evidence that the latter may be a key part of the pathology of chronic neurodegenerative disorders such as Huntington's and Alzheimer's diseases (for review, see Ref. 15). We recently provided evidence that apoptosis may play an important role in the pathophysiology of degenerated tendons (39). It is therefore a possibility that overexcitation of these signaling mechanisms is a cause of the degeneration that characterizes tendinopathy.

To test whether glutamate could elicit a similar excitotoxic response in tendon cells, rat supraspinatus fibroblasts were cultured and exposed to glutamate for 24 h, and apoptotic cell death was assayed by colorimetric TUNEL and caspase-3 (DEVDase) protease activity assays. Both TUNEL staining (Fig. 4) and caspase-3 activity assays (Fig. 5) showed a significant increase in apoptotic cells after exposure to glutamate compared with unstimulated controls, with levels similar to that of hydrogen peroxide-treated (positive control) cells. This apoptotic response could also be decreased by the potent, irreversible, and cell-permeable pan-caspase inhibitor z-VAD-fmk. This is in agreement with a study by May and Gray (22), in which moderate levels of glutamate caused rapid degeneration and death in skin fibroblasts, and also more recently in a study by Steinmetz et al. (34) that demonstrated death of mouse fibroblasts within 1 h of glutamate treatment.

Stress response gene activation in tendinopathic tendon.   Also of interest was the presence of several stress genes related to excitotoxicity and apoptosis among the most up- and downregulated genes, including HSP27, HST70, and FLIP, which may indicate that tendon cells were responding to the insults that cause apoptosis by attempting to regulate apoptotic pathways. Both HSP27 and the HST70 homolog HSP70 are stimulated by and/or have protective effects on neuronal cells undergoing glutamate excitotoxicity (16, 28), and whereas FLIP has not previously been associated with glutamate excitotoxicity, it is an important regulator of caspase activity. Although the expression of neither HSP27 nor HST70 could be regulated by glutamate in vitro, FLIP showed a small but significant increase in expression that could be returned to near-control levels by pretreatment of the cells with MK801. The altered expression of these anti-apoptotic stress genes in degenerated tendon and their direct regulation by glutamate in tendon fibroblasts in vitro may suggest they have some role in minimizing the effect of excitotoxicity within the tendinopathic tendon.

In conclusion, we documented a range of gene expression in the degenerated rat supraspinatus tendon, specifically identifying members of CNS-like glutamate signaling cascades as being significantly differentially expressed in this tissue. It has been hypothesized that osteoblasts use a mechanism similar to long-term potentiation in neurons of the CNS to instigate long-term structural changes of the surrounding bone in response to short-term mechanical stresses, and the same may hold true for their upregulation in the tendinopathic tendon. If this proves to be the case, then it is a further possibility that overexcitation could be a cause of the degeneration of tendon tissue seen in tendinopathy. Significantly high levels of intratendinous glutamate have been observed in several in vivo tendinopathy studies, both animal and human, although thus far no satisfactory explanation has been put forward for its presence (1, 2). Glutamate was shown here both to trigger apoptosis in cultured tendon fibroblasts and directly regulate the expression of apoptosis-related genes. This regulation of gene expression could be prevented by the pretreatment of tendon cells with the glutamate receptor antagonist MK801. To the authors' knowledge, this is the first time these effects have been linked to the tendon cells or shown to exist in any connective tissue pathology. If glutamate does indeed prove to play an important role in tendon degeneration, future work could include testing some of the well-characterized, clinically tested NMDA antagonists (such as Memantine, which is currently used as a treatment for dementia) in a tendinopathy model. There are several effective, well-tolerated blockers of sustained glutamate receptor activation, and the pharmacological properties of some prevent them crossing the blood-brain barrier, lessening the chance of unwanted effects on the brain. Studies such as these will be necessary to help formulate effective therapies where currently none exist for the management of this extremely common and debilitating disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by St. George Hospital/South East Area Health Service.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Bronwyn Robertson and the Ramaciotti Centre at the University of New South Wales for supplying the microarrays and for invaluable advice and Chris Molloy for the development of the Combina.tions.net search tool.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. A. C. Murrell, Level 2 Research and Education Bldg., 4–10 South St, Kogarah, NSW 2217, Australia (E-mail: admin{at}ori.org.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Alfredson H, Ljung BO, Thorsen K, and Lorentzon R. In vivo investigation of ECRB tendons with microdialysis technique—no signs of inflammation but high amounts of glutamate in tennis elbow. Acta Orthop Scand 71: 475–479, 2000.[CrossRef][ISI][Medline]
  2. Alfredson H, Thorsen K, and Lorentzon R. In situ microdialysis in tendon tissue: high levels of glutamate, but not prostaglandin E2 in chronic Achilles tendon pain. Knee Surg Sports Traumatol Arthrosc 7: 378–381, 1999.[CrossRef][ISI][Medline]
  3. Almekinders LC, Banes AJ, and Ballenger CA. Effects of repetitive motion on human fibroblasts. Med Sci Sports Exerc 25: 603–607, 1993.
  4. Almekinders LC, Baynes AJ, and Bracey LW. An in vitro investigation into the effects of repetitive motion and nonsteroidal antiinflammatory medication on human tendon fibroblasts. Am J Sports Med 23: 119–123, 1995.[Abstract/Free Full Text]
  5. Bhangu PS, Genever PG, Spencer GJ, Grewal TS, and Skerry TM. Evidence for targeted vesicular glutamate exocytosis in osteoblasts. Bone 29: 16–23, 2001.[Medline]
  6. Brustovetsky N, Brustovetsky T, Jemmerson R, and Dubinsky JM. Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane. J Neurochem 80: 207–218, 2002.[CrossRef][ISI][Medline]
  7. Danbolt NC. Glutamate uptake. Prog Neurobiol 65: 1–105, 2001.[CrossRef][ISI][Medline]
  8. Dong H, Zhang P, Song I, Petralia RS, Liao D, and Huganir RL. Characterization of the glutamate receptor-interacting proteins GRIP1 and GRIP2. J Neurosci 19: 6930–6941, 1999.[Abstract/Free Full Text]
  9. Downing DS and Weinstein A. Ultrasound therapy of subacromial bursitis. A double blind trial. Phys Ther 66: 194–199, 1986.[ISI][Medline]
  10. Green ES, Rendahl KG, Zhou S, Ladner M, Coyne M, Srivastava R, Manning WC, and Flannery JG. Two animal models of retinal degeneration are rescued by recombinant adeno-associated virus-mediated production of FGF-5 and FGF-18. Mol Ther 3: 507–515, 2001.[CrossRef][ISI][Medline]
  11. Gu Y, Genever PG, Skerry TM, and Publicover SJ. The NMDA type glutamate receptors expressed by primary rat osteoblasts have the same electrophysiological characteristics as neuronal receptors. Calcif Tissue Int 70: 194–203, 2002.[CrossRef][ISI][Medline]
  12. Gui Y and Murphy LJ. Interaction of insulin-like growth factor binding protein-3 with latent transforming growth factor-beta binding protein-1. Mol Cell Biochem 250: 189–195, 2003.[CrossRef][ISI][Medline]
  13. Hinoi E, Fujimori S, and Yoneda Y. Modulation of cellular differentiation by N-methyl-D-aspartate receptors in osteoblasts. FASEB J 17: 1532–1534, 2003.[Abstract/Free Full Text]
  14. Hsu RW, Hsu WH, Tai CL, and Lee KF. Effect of hyperbaric oxygen therapy on patellar tendinopathy in a rabbit model. J Trauma 57: 1060–1064, 2004.[ISI][Medline]
  15. Hynd MR, Scott HL, and Dodd PR. Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease. Neurochem Int 45: 583–595, 2004.[CrossRef][ISI][Medline]
  16. Kalwy SA, Akbar MT, Coffin RS, de Belleroche J, and Latchman DS. Heat shock protein 27 delivered via a herpes simplex virus vector can protect neurons of the hippocampus against kainic-acid-induced cell loss. Brain Res Mol Brain Res 111: 91–103, 2003.[Medline]
  17. Kannus P. Etiology and pathophysiology of chronic tendon disorders in sports. Scand J Med Sci Sports 7: 78–85, 1997.[ISI][Medline]
  18. Kannus P and Jozsa L. Histopathological changes preceding spontaneous rupture of a tendon. A controlled study of 891 patients. J Bone Joint Surg Am 73: 1507–1525, 1991.[Abstract/Free Full Text]
  19. Kiessling S, Muller-Newen G, Leeb SN, Hausmann M, Rath HC, Strater J, Spottl T, Schlottmann K, Grossmann J, Montero-Julian FA, Scholmerich J, Andus T, Buschauer A, Heinrich PC, and Rogler G. Functional expression of the interleukin-11 receptor alpha-chain and evidence of antiapoptotic effects in human colonic epithelial cells. J Biol Chem 279: 10304–10315, 2004.[Abstract/Free Full Text]
  20. Li Z, Yang G, Khan M, Stone D, Woo SL, and Wang JH. Inflammatory response of human tendon fibroblasts to cyclic mechanical stretching. Am J Sports Med 32: 435–440, 2004.[Abstract/Free Full Text]
  21. Mason DJ, Suva LJ, Genever PG, Patton AJ, Steuckle S, Hillam RA, and Skerry TM. Mechanically regulated expression of a neural glutamate transporter in bone: a role for excitatory amino acids as osteotropic agents? Bone 20: 199–205, 1997.[Medline]
  22. May PC and Gray PN. The mechanism of glutamate-induced degeneration of cultured Huntington's disease and control fibroblasts. J Neurol Sci 70: 101–112, 1985.[CrossRef][ISI][Medline]
  23. McLauchlan GJ and Handoll HH. Interventions for treating acute and chronic Achilles tendinitis. Cochrane Database Syst Rev CD000232, 2001.
  24. Molloy TJ, Wang Y, Horner A, Skerry TM, and Murrell GA. Microarray analysis of healing rat Achilles tendon: evidence for glutamate signaling mechanisms and embryonic gene expression in healing tendon tissue. J Orthop Res 24: 842–855, 2006.[CrossRef][ISI][Medline]
  25. Nicholls DG. Mitochondrial dysfunction and glutamate excitotoxicity studied in primary neuronal cultures. Curr Mol Med 4: 149–177, 2004.[CrossRef][ISI][Medline]
  26. Patton AJ, Genever PG, Birch MA, Suva LJ, and Skerry TM. Expression of an N-methyl-D-aspartate-type receptor by human and rat osteoblasts and osteoclasts suggests a novel glutamate signaling pathway in bone. Bone 22: 645–649, 1998.[Medline]
  27. Rothman SM. The neurotoxicity of excitatory amino acids is produced by passive chloride influx. J Neurosci 5: 1483–1489, 1985.[Abstract]
  28. Sharp JW. PCP and ketamine inhibit non-NMDA glutamate receptor mediated hsp70 induction. Brain Res 728: 215–224, 1996.[CrossRef][ISI][Medline]
  29. Skerry TM. Identification of novel signaling pathways during functional adaptation of the skeleton to mechanical loading: the role of glutamate as a paracrine signaling agent in the skeleton. J Bone Miner Metab 17: 66–70, 1999.[CrossRef][ISI][Medline]
  30. Sommerich CM, McGlothlin JD, and Marras WS. Occupational risk factors associated with soft tissue disorders of the shoulder: a review of recent investigations in the literature. Ergonomics 36: 697–717, 1993.[Medline]
  31. Soslowsky LJ, Thomopoulos S, Tun S, Flanagan CL, Keefer CC, Mastaw J, and Carpenter JE. Neer Award 1999. Overuse activity injures the supraspinatus tendon in an animal model: a histologic and biomechanical study. J Shoulder Elbow Surg 9: 79–84, 2000.[CrossRef][ISI][Medline]
  32. Spencer GJ and Genever PG. Long-term potentiation in bone—a role for glutamate in strain-induced cellular memory (Abstract)? BMC Cell Biol 4: 9, 2003.[CrossRef][Medline]
  33. Stapelberg M, Tomasetti M, Alleva R, Gellert N, Procopio A, and Neuzil J. alpha-Tocopheryl succinate inhibits proliferation of mesothelioma cells by selective down-regulation of fibroblast growth factor receptors. Biochem Biophys Res Commun 318: 636–641, 2004.[CrossRef][ISI][Medline]
  34. Steinmetz RD, Fava E, Nicotera P, and Steinhilber D. A simple cell line based in vitro test system for N-methyl-D-aspartate (NMDA) receptor ligands. J Neurosci Methods 113: 99–110, 2002.[CrossRef][ISI][Medline]
  35. Triantafillopoulos IK, Banes AJ, Bowman KF Jr, Maloney M, Garrett WE Jr, and Karas SG. Nandrolone decanoate and load increase remodeling and strength in human supraspinatus bioartificial tendons. Am J Sports Med 32: 934–943, 2004.[Abstract/Free Full Text]
  36. Uhthoff HK and Sano H. Pathology of failure of the rotator cuff tendon. Orthop Clin North Am 28: 31–41, 1997.[CrossRef][ISI][Medline]
  37. Wiley P. Low energy extracorporeal shock-wave treatment for tendinitis of the supraspinatus (Abstract). Clin J Sport Med 12: 262, 2002.[CrossRef][Medline]
  38. Yuan J, Murrell GA, Trickett A, and Wang MX. Involvement of cytochrome c release and caspase-3 activation in the oxidative stress-induced apoptosis in human tendon fibroblasts. Biochim Biophys Acta 1641: 35–41, 2003.[Medline]
  39. Yuan J, Wang MX, and Murrell GA. Cell death and tendinopathy. Clin Sports Med 22: 693–701, 2003.[CrossRef][ISI][Medline]



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