VGluT2 expression in painful Achilles and patellar tendinosis: Evidence of local glutamate release...

VGluT2 Expression in Painful Achilles and Patellar Tendinosis:Evidence of Local Glutamate Release by Tenocytes

Alexander Scott,1,2 Hakan Alfredson,1 Sture Forsgren2

1Department of Surgical and Perioperative Science, Umea University, Sweden

2Department of Integrative Medical Biology, Umea University, Sweden

Received 25 May 2007; accepted 24 August 2007

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20536

ABSTRACT: The pathogenesis of chronic tendinopathy is unclear. We have previously measuredhigh intratendinous levels of glutamate in patients with tendinosis, suggesting potential roles ofglutamate in the modulation of pain, vascular function, and degenerative changes includingapoptosis of tenocytes. However, the origin of free glutamate found in tendon tissue is completelyunknown. Surgical biopsies of pain-free normal tendons and tendinosis tendons (Achilles andpatellar) were examined immunohistochemically using antibodies against vesicular glutamatetransporters (VGluT1 and VGluT2), as indirect markers of glutamate release. In situ hybridizationfor VGluT2mRNAwas also conducted. Specific immunoreactions for VGluT2, but notVGluT1, couldbe consistently detected in tenocytes. However, therewere interindividual variations in the levels ofimmunoreactivity. The level of immunoreaction for VGluT2 was higher in tendinosis tendonscompared to normal tendons (p<0.05). In situ hybridization of VGluT2 demonstrated that mRNAwas localized in a similar pattern as the protein, with marked expression by certain tenocytes,particularly those showing abnormal appearances. Reactivity for VGluT1 and -2 was absent fromnerves and vessel structures in bothnormal and painful tendons. The current data demonstrate thattenocytesmaybe involved in the regulationof extracellular glutamate levels in tendons. Specifically,the observations suggest that free glutamate may be locally produced and released by tenocytes,rather than by peripheral neurons. Excessive free glutamate is expected to impact a variety ofautocrine and paracrine functions important in the development of tendinosis, including tenocyteproliferation and apoptosis, extracellular matrix metabolism, nociception, and blood flow. � 2007

Orthopaedic Research Society. Published byWiley Periodicals, Inc. J Orthop Res 26:685–692, 2008

Keywords: tendinosis; Achilles; patellar; tendon; glutamate

INTRODUCTION

Glutamate is a pervasive amino acid with a well-known role in excitatory synaptic transmissionin the central nervous system (CNS), where it isconsidered responsible for transmitting most fastsynaptic potentials.1,2 The transmission of cellularsignals among neurons by glutamate requires anumber of genes including dedicated glutamatereceptors, receptor interacting proteins, plasmamembrane transporters, and vesicular transport-ers.3 There are two main classes of glutamatereceptors, including metabotropic receptors (sig-naling via IP3, diacylglycerol, and cyclic AMP) andionotropic receptors (capable of altering perme-ability to specific cations). Ionotropic receptors arefurther classed as N-methyl-D-aspartate (NMDA)receptors, DL-a-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors, and kainate (KA)

receptors.3 In addition to receptors, glutamatetransporters in the plasma membrane (GLAST,GLT-1, EAAC1, EAAT4, -5) or in vesicles (VGluT1-3) play key roles in the regulation of extracellularglutamate concentrations.4

It has become increasingly evident that genesencoding elements of the glutamate signalingmachinery are also expressed by non-neuronalcells in a variety of peripheral tissues, and thatglutamate functions as a regulatory cytokine withautocrine and paracrine functions in diverse phy-siologic processes such as bone turnover, insulinand growth hormone secretion, platelet formationand function, and keratinocyte development anddifferentiation.1,5,6 In addition to diverse roles inphysiologic cell signaling, glutamate also playsknown or emerging roles in a range of pathologiesincluding inflammation and soft tissue repair.7–11

During acute inflammation of the knee, glutamateis released into the knee joint from peripheralglutamatergic nerves, resulting in a nitric oxide–dependent increase in local blood flow.8 Further-more, glutamate receptor antagonism can reduce

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Correspondence to: Sture Forsgren (Telephone: þ 46 (0) 90786 5147; E-mail: [email protected])

� 2007 Orthopaedic Research Society. Published by Wiley Periodicals,Inc.

nociception and sensitization in response to avariety of inflammatory stimuli.12

Painful overuse tendon injuries are a commonmedical problem seen by general and sports medi-cine practitioners, with a cumulative incidence of52% in middle- or long-distance runners (Achillestendinopathy) and a prevalence of 32% to 44% withcurrentsymptoms inelite jumpingathletes (patellartendinopathy).13,14 The pathology considered to beresponsible for many cases of chronic tendon over-use pain is often classified as tendinosis. Tendinosisis characterized morphologically by collagen frag-mentation and disarray, tenocyte abnormalities(including apoptosis and necrosis, increasedproliferation, and excessive glycosoaminoglycanproduction) and by an increase in local intra- andperitendinous blood flow.15,16 In this condition, highlevels of glutamatehavebeenmeasured fromwithintendinosis lesions using microdialysis in patientswith Achilles, patellar, and extensor carpi radialisbrevis tendinosis.17–19 The observations thus sug-gest thatglutamatemaycontribute to the symptomsof chronic tendon pain.17–19 Glutamate has alsorecently been suggested by Murrell and coworkersto be present within tendinosis lesions at levelssufficiently high to induce tenocyte apoptosis,20 apathological feature of some advanced tendinosislesions.21,22

Beyond the high levels of glutamate detected bymicrodialysis in painful tendons, nothing is knownregarding the glutamate signaling machinery thatis present innormal or pathological human tendons,although we previously reported that NMDA recep-tors are present in nerve fascicles in the humanAchilles and patellar tendons.17,23 More recentstudies conducted on rat tendons have documentedthe presence of mRNA in tenocytes for several keyelements of glutamate signaling machinery, includ-ing receptors (metabotropic glutamate receptorsmGluR5 and mGluR6, NMDARL1), glutamatereceptor–interacting proteins (GRIP1 and GRIP2),and plasma membrane transporters (EAAT4).20,24

To our knowledge, there is no information onelements of the signal transduction pathwayrequired for vesicular release of glutamate inhuman tendons, namely the presence of vesicularglutamate transporters, either at the protein ormRNA levels. This lack of knowledge is a drawbackwhen considering the fact that high extracellularglutamate levels occur in tendinosis. In contrast toplasma membrane transporters that play a role insignal termination by internalization of glutamate,VGluTs are required for the transport of glutamateinto secretory vesicles, and may therefore be con-sidered as indirect markers of potential sources of

free glutamate. VGluT1 and 2 are widely expressedin the CNS and considered definitive markers forexcitatoryglutamatergic synapses,whereasVGluT3has a more restricted pattern of CNS expression.25

Therefore, the purpose of the current studywas to determinewhetherVGluT1 or -2 are presentin human tendons. Further, our purpose wasto determine whether their expression levels orpattern of distribution would be influenced bytendinosis pathology. We hypothesized that atleast one or more of the two main VGluT isoformsexamined would be present to a greater degree intendinosis, due to the higher levels of extracellularglutamate associated with this condition.

METHODS

Patients and Tendon Sampling

The current study included samples from 29 individuals(30 patellar or Achilles tendons). There were 14 tendi-nosis patients (1 patellar, 13 Achilles) and 15 subjectswith no history of current or past tendon pain (8 patellarand 7 Achilles). There were 19 men and 10 women,average age 40.7 years (range, 18–54 years), with nosignificant difference in age between patient and controlgroups. Both Achilles and patellar tendons wereincluded, as it has been reported that there isno significant difference in tendon pathology betweenthe two sites, that is, the morphology seen in patellartendinosis closely resembles that seen in Achillestendinosis.26 All tendinosis patients displayed tender-ness and pain during loading, and exhibited tendonchanges verified by ultrasonography (localized wideningof the tendon, irregular structure, and focal hypoechoicareas) or magnetic resonance imaging (MRI; increasedsignal intensity and localized widening). All subjectswere otherwise healthy and on no medications.

In the case of tendinosis patients, samples (2–3mm3 insize) were obtained from the area of the tendon corre-sponding to structural changes observed on ultrasound orduring surgery. Sampleswere transported on ice in sterileconditions to the laboratory. Some samples were fixedovernight in4%formaldehydebuffered in0.1Mphosphatebuffer at 48C, followed by embedding in OCT (MilesLaboratories, Naperville, IL) and freezing. Other sampleswere directly snap frozen in isopentane chilled in liquidnitrogen. All samples were then stored at �808.

The study protocol was approved by the Committee ofEthics at the Faculty of Medicine and Odontology, UmeaUniversity, and the Regional Ethical Review Board inUmea, and the experiments were conducted according tothe principles expressed in the Declaration of Helsinki.

Morphological Analysis

The presence of any significant artifacts was examinedin longitudinal haematoxylin–eosin (H&E) sectionsfrom normal and painful tendons using previously

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published morphological features of tendinosis.27 Allsamples included were free from significant artifact,and the tendinosis samples demonstrated, to varyingdegrees, typical features of the pathology includingtenocyte hypercellularity, pyknotic or necrotic nuclei,tenocyte rounding, vascular hyperplasia, and collagenfibre disarray.

Immunofluorescence for VGluT1 and -2

Immunohistochemical staining was carried out on biop-sies from all patients using goat polyclonal antibodiesobtained from Santa Cruz Biotechnology (Santa Cruz,CA), namely sc26026 and sc46569 for VGluT1 and -2respectively. Control staining included omission of theprimary antibody, replacement of primary antibody withnormal goat serum, or pre-absorption of the antibodywith the corresponding blocking peptide at 150 to 200 mg/mL (sc26026P for VGluT1 and sc46569P for VGluT2).Both fixed and unfixed samples were included, as fixationappeared to have a minimal influence on the strengthor distribution of observed reactions for VGluT1 or-2.Specimens of fixed and unfixed human colitis tissue wereused as a reference tissue.28 After air drying for 30min atroom temperature, 7-mm thick crysections were treatedwith acid potassium permanganate to reduce fixation-induced autofluorescence (in fixed samples), then washedin phosphate-buffered saline (PBS) and placed in 1%Triton in PBS for 20 min, rinsed in PBS, and blocked for15 min with normal 5% donkey serum. Primary anti-bodies were applied overnight at 48C at 1:100 dilution,rinsed in PBS, then incubated with the secondary anti-body (1:100 donkey antigoat conjugated to FITC; JacksonImmunoResearch Europe, Newmarket, UK) for 30min inthe dark. Sections were washed and coverslipped withVectashield hardset mounting medium (Vector Labora-tories, Burlingame, CA) and viewed with a fluorescencemicroscope (Axioskop2, Carl Zeiss, Stockholm, Sweden)to determine the location and intensity of specificreactions. All sections, including those with and withoutpeptide block, were viewed independently and thentogether by two observers (AS and SF) to achieve con-sensus regarding the extent and specificity of reactions.Parallel sections of some biopsies were stained identicallyon separate occasions to ensure repeatability of theprocedure, and each round of staining included bothnormal and tendinosis tendons to ensure validity ofcomparisons.

In Situ Hybridization for VGLUT-2

In situ hybridization was carried out on a subset oftendon samples (n¼ 3) to examine whether the mRNAwould be localized in a similar or different pattern tothe protein. A digoxigenin (DIG)-hyperlabeled oligonu-cleotide probe (ssDNA) for detection of human VGluT2mRNA was used on sections from frozen tendinosisspecimens. The probe sequence (CCTTG TACAAATTCC TCTTT CTTTT CCCAA CCACT AGGCCAACCT CCA) was complementary to nucleotides 2066

to 2133 located within the coding sequence of humanVGluT2 (GeneDetect, Auckland, New Zealand). In situhybridization was performed according to an establishedprotocol,29 using an alkaline phosphatase (AP)-labeledanti-DIG antibody (GeneDetect) for detection. A seriesof 10-mm thick cryosections were air-dried at roomtemperature (RT) for 30 min, then fixed in sterile 4%paraformaldehyde (PFA) in 0.1M PB for 60 min at RT.The slides were then washed with 2� saline sodiumcitrate (SSC) for 2� 10 min. The sections were there-after incubated in 0.2M HCl for 8 min at RT to inhibitendogenous alkaline phosphatase activity. After this,the sections were acetylated by incubation of slides for15 min at RT in a mixture of 195 mL DEPC-H2O, 2.7 mLtiethanolamine, 0.355 mL HCl, and 0.5 mL aceticanhydride. Slides were then again rinsed in 2�SSC.After that, 50 ng of the ssDNA probe was put in 15 mL ofhybridization solution in a 1.5-mL Eppendorf tube,denaturated for 5 min at 808C, and then put on ice.The probe-containing hybridization solution [500 mLformamide, 200 mL 20�SSC, 50 mL of 20� Denhardt’ssolution, 50 mL herring sperm DNA (10 mg/mL) heat-denatured, 25 mL bakers yeast RNA (10 mg/mL),175 mL dextran sulfate (50%)] was then applied toeach section and incubated at 568C overnight. The slideswere then washed for 2� 10 min at RT in 2�SSC andfor 5 min at RT in STE-buffer [STE-buffer: 500 mMNaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA] andincubated in 100 mL RNase A (40 mg/mL in STE) for30 min at 378C. After this, the slides were washed for20 min at 568C in 2�SSC, 50% formamide (25 mL 100%and 25 mL 2�SSC buffer), then placed for 2� 5 min atRT in 1�SSC, and for 2� 5 min at RT in 0.5�SSC.Then the slides were washed for 5 min in buffer 1[100 mM Tris-HCl (pH 7.5)þ 150 mM NaCl) andincubated in buffer 1 containing 4% normal horse serum(NHS) for 60 min at RT in a humid chamber. Sectionswere then incubated in 100 mL of the AP-labeled anti-DIG antibody (diluted 1:500 in buffer 1 with 4% NHS)for 60 min at RT in a humid chamber. The slides werethen washed for 2� 10 min in buffer 1, and for 2� 5 minin buffer 2 [100 mM Tris-HCl (pH 9.5)þ 100 mMNaClþ 50 mM MgCl2]. After this, the enzyme (AP)substrate solution (20 mL NBT/BCIP in 1 mL buffer 2with 10 mL levamisole) was sterile filtered (22 mm) andadded to the sections, and slides were incubated upsidedown in the dark at 48C overnight. Color reaction wasstopped by placing the slides in buffer 3 [10 mM Tris-HCl (pH 8.0)þ 1 mM EDTA]. Slides were then de-hydrated and counterstained in 0.5% methyl green andmounted in Pertex microscopy mounting medium. Thecorresponding sense DIG-hyperlabeled ssDNA probeswere used as negative controls. As positive control, ab-actin probe (GD5000-OP) was used (GeneDetect).

Statistical Analysis

The extent of immunofluorescence was graded semi-quantitatively from 0 (absent expression) to 3 (intenseand widespread expression) concerning specific tenocyte

EVIDENCE OF GLUTAMATE RELEASE BY TENOCYTES 687


immunofluorescence, and a Mann–Whitney U test wasconducted on the resultant data (n¼ 30) with statisticalsignificance predetermined at p¼ 0.05.

RESULTS

Immunofluorescence

Specific VGluT1 immunofluorescence could not beobserved in either tendon or colon. By contrast,VGluT2 immunofluorescence was observed in bothtendon and colon. VGluT2 immunoreactions werenot present in tissue exposed to the preabsorbedantibody processed in parallel at the same concen-tration (Fig. 1). In tendon, VGluT2 expression wasobserved in tenocytes and not in other structures(nerves or blood vessels). In colon specimens,VGluT2 expression was detected in the myentericplexus, as previously described.28

VGluT2 immunoreaction was detected in 38% ofnormal tendons and in 71% of painful tendons.Accordingly, semiquantitative grading revealeda significantly greater expression of VGluT2 intenocytes from tendinosis patients than in those

of controls (p¼ 0.005, Fig. 2). The expression ofVGluT2 in tendinosis tendons was thus both morewidespread (i.e., large numbers of tendons showedimmunoreactive tenocytes) and more intense thanin normal tendons (Fig. 3).

VGluT2 immunoreactivitity in tenocytes wasoften seen in a punctate cytoplasmic pattern(Fig. 1). Reactivity was most intense in abnormallyappearing tenocytes, including those showingrounded (as opposed to spindle shaped) or crimped(i.e., wavy) appearances (Fig. 3A). VGluT2 immu-noreactivity in control tendons showed a similarpattern, but was often less intense and less wide-spread (Fig. 3B).

Figure 1. VGluT2 immunofluorescence in tendinosis tendon.(A) Numerous tenocytes demonstrating positive immunoreac-tions (original magnification,�40). (B) Adjacent section demon-strating no reactions with preabsorbed antibody.

Figure 2. Results of VGluT2 immunofluorescence in teno-cytes using semiquantitative grading. *Significantly different,p¼0.005 (Mann–Whitney U test). Error bars represent stand-ard deviation. 0, no immunoreaction; 3, intense and widespreadimmunoreaction.

Figure 3. Marked VGluT2 immunofluorescence in abnormaltenocytes. (A) Many tenocytes with an exaggerated crimp(waviness) are present in regions of tendinosis samples.Rounded tenocytes (arrows) are also present in the same field.Inset: Higher power view of a rounded tenocyte, demonstratingevident cytoplasmic localization of VGluT2. (B) Tenocytesin healthy tendon, demonstrating faint punctate reactions.

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Therewasno apparent difference in the extent ofexpression amongmenorwomen,noranyapparentrelationship with age.

VGluT2 mRNA in Tenocytes

VGluT2 mRNA expression was observed in themajority of tenocytes in both normal and abnormalregions of tendon, suggesting a more sensitivedetection of VGluT2 mRNA compared to VGluT2immunofluorescence (Fig. 4). Nevertheless, theexpression levels were somewhat variable withinand between specimens, being most marked intenocytes with abnormal appearance (rounded,broadened, or crimped). Expression was not seenin any nerve or blood vessel structures. Parallel insitu hybridization with the sense (control) probeverified that the observed AP end-reaction productresulted from the specific hybridization of theDNA probe to its complementary RNA sequencerather than due to a non-specific interaction(Fig. 4B, inset). The b-actin probe labeled bothtenocytes and vascular cells.

DISCUSSION

The current study documents the presence ofvesicular glutamate transporters (VGluT2) in a

novel cell type—the tendon fibroblast (tenocyte).This is completely new information for humantendons. However, along with other elementsof glutamate signaling machinery which haverecently been reported in rat tenocytes (glutamatereceptors, glutamate receptor interacting pro-teins, and plasma membrane transporters),20,24

the current report concerning VGluT2 mRNA andprotein supports a hypothesis that a peripheralglutamatergic system may be present in tendons.Moreover, the expression of VGluT2 at the proteinlevel was most prominent in patients with chronictendon pain (p¼ 0.005), which strengthens pre-vious suggestions that glutamate may be involvedin the pathology of tendinosis.17,23

Non-Neuronal Expression of aGlutamatergic Marker in Tendons

In the CNS, VGluT1 and -2 are expressed specifi-cally at excitatory, glutamatergic synapses andplay key roles in synaptogenesis as well as inmemory and learning.2,3 In the peripheral nervoussystem, the expression of vesicular glutamatetransporters has been documented at a variety oflocations including nerve ganglia in the colonicmucosa, the extrinsic and intrinsic innervation ofthe rat esophagus, in nerve terminals contactingneuroepithelial bodies in the lung, and in freenerve endings in the palatine mucosa.28,30,31

Expression has also recently been revealed inmechanosensitive nerve-associated structuressuch asMerkel cells andmuscle spindles, implyingthat glutamate signaling in these locations mayplay a role in modulating touch sensitivity andmuscle tone.32,33 It is therefore of interest to notethat in the present study, VGluT1 or -2 expressionwas not observed in nerve elements. Duringinflammation of joint structures (i.e., the ratknee synovium), glutamate has been suggestedto be locally released by glutamatergic nerves,as the increase in glutamate could be blocked byblockade of peripheral nerves with lidocaine ordorsal rhizotomy.8 Conversely, the current studysuggests that free glutamate within painful ten-dons probably derives from locally located cells(the tenocytes), particularly in tendinosis tendons.

Evidence for Peripheral Glutamatergic Cell Signaling

VGluTs have been proposed as molecular markersthat may define the existence of a peripheral,locally acting glutamatergic signaling system withautocrine and paracrine functions. In the pinealgland, VGluT1 and -2 are expressed in synapticlikemicrovessels, and functional glutamate signaling

Figure 4. VGluT2 in situ hybridization. (A) VGluT2 expres-sion shows variability, with some tenocytes showing markedexpression (arrows) and others only faint or absent (asterisks).(B, C) Abnormal (rounded or broadened) tenocytes demonstrat-ing prominent expression of VGluT2 (arrows), with closelyadjacent tenocytes demonstrating minimal or no expression(asterisks). Inset: Sense control in a section adjacent to (B)demonstrates only background reactions.



(via metabotropic receptors) was shown to beinvolved in an important pineal gland function,the regulation of melatonin synthesis.1 VGluT1 and-2 are also expressed in glucagon-containing secre-tory granules in a cells in the Islets of Langherhans,where neighboring b cells express functionalAMPA-type receptors.34 Even more establishedis a physiologic role of glutamate signaling in theregulation of bone turnover, a process regulated inpart bymechanotransduction (the cellular responseto mechanical force). In bone, VGluT1 is expressedby mature osteoclasts and is directly responsiblefor their ability to release glutamate via trans-cytotic vesicles.35 Furthermore, VGluT1 knockoutmice develop an osteoporotic phenotype, strength-ening the suggestion that glutamate plays a keyrole in suppressing bone resorption.35 Another bonecell type, osteoblasts, do not express VGluTs35 butdemonstrate all classes of glutamate receptors aswell as plasma membrane transporters (reviewedin Chenu36). The expression levels of severalionotropic and metabotropic glutamate receptorson osteoblasts was shown to be reduced bymechanical loading of bone segments in vitro.37

Likewise, in osteocytes and osteoblasts, GLASTexpression appears to be reduced in areas of load-induced bone formation in vivo.38 Thus, paracrineglutamate signaling amongst osteoclasts, osteo-blasts, and osteocytes is heavily implicated in theprocess of mechanotransduction in bone. Prelimi-nary evidence also points to a requirement forglutamate signaling in chondrocyte mechanotrans-duction.39,40

Potential Role of Glutamate in TendonMechanotransduction

Like bone and cartilage, tendon is a tissue in whichthe content and structure of the extracellularmatrix is dynamically regulated in response toaltered loading conditions. Mechanotransductionin tendon is relatively understudied, however.Tenocytes form elaborate gap-junction–linkedarrays and, in response to mechanical load, releasea variety of cytokines that are thought to regulatethe activation of calcium-dependent signal path-ways leading to proliferation and increased turn-over and production of the collagen–proteoglycanextracellular matrix.41 Like chondrocytes andosteoblasts, human tenocytes express both voltage-gated calcium channels as well as the mechanosen-sitive tandem pore domain potassium channelTREK-1.42 Considering that the hyperpolarizationresponse to mechanical stimulation in chondrocytesrequires NMDA receptor activation,39 the current

study supports the hypothesis that tendons mayexhibit a similar dependence on glutamate signal-ing for certain mechanotransduction signalingevents. Thus, the regulation of extracellular gluta-mate concentrations by tenocytes via VGlut2and via plasma membrane transporters requiresfurther study.

Role of Glutamate in Tendinopathy

Current evidence from physiologic studies inhumans and animals suggests that activationof AMPA and NMDA receptors can result inperipheral nociception.9,10,12,43 We have previ-ously shown that peripheral nerves in humantendons demonstrate the existence of NMDAreceptors, which have also been observed in thenerve fibers of skin.44 A suggestion that is moredirectly supported by the current observations isthat glutamate could modulate tenocyte behaviorwithin the tendinosis lesion. Glutamate is a well-known toxic stimulus when present in excess,leading to necrosis or apoptosis (or both) in avariety of cell types including neurons, fibroblasts,and chondrocytes.20,45,46 Murrell and coworkershave demonstrated that primary tenocyte cellcultures undergo a significant increase in the rateof cell death when exposed to 500 mM glutamate for24 h. A strength in the study by Murrell et al. isthat the glutamate concentration to which teno-cytes were exposed is on the same scale as hasbeen measured in tendinpathy patients withmicrodialysis (250 mM).19,20 Advanced tendinosislesions (ruptured supraspinatus tendon or long-standing severe jumper’s knee) also demonstrateincreased numbers of apoptotic and necrotictenocytes.16,21,22 Therefore, increased glutamatelevels may contribute to cell death and subsequentlocal tissue degeneration in tendinopathy. Largenumbers of apoptotic tenocytes have been reportedin equine tendinosis lesions, but relatively few inthe standard rat laboratory model, making it achallenge to study potential roles of glutamate andits relation to apoptosis in tendon overuseinjury.47,48 Glutamate toxicity and cell death arealso thought to play a role in disorders such asAlzheimer’s disease.49

Although tenocyte death occurs in some tendi-nosis lesions, chronically painful tendons are ingeneral hypercellular mainly due to increasednumbers of tenocytes, with a variety of otherreparative cell types also found.50 Thus, anotherhighly relevant feature of tendinosis pathology thatglutamate could influence is cellular proliferation.Glutamate NMDA and AMPA antagonists inhibit

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cellular proliferation in a variety of studied cellcultures, including human colon adenocarcinoma,astrocytoma, breast and lung carcinoma, andneuroblastoma.21 Furthermore, the addition of500 mM glutamate to synovial fibroblasts resultedin a doubling of BrdU incorporation.11 Interest-ingly, this stimulation of fibroblast proliferation byglutamate only occurred in fibroblast cultures thathad been derived from arthritic animals (collagen-induced rat arthritis model), suggesting that theinfluence of glutamate on cell behavior involves as-yet unspecified interactions with other molecularaspects of the pathology. Nonetheless, elevatedglutamate levels in tendinosis lesions could poten-tially contribute to the simultaneous presence ofincreased apoptosis, necrosis, and proliferationthat is observed in tendinosis.16 Glutamate mayalso modulate pro-apoptotic or proliferative effectsof other conditions thought to be present in tendon,including hypoxia or oxidative stress.

In conclusion, the current study demonstratesthe existence of VGluT2 in human tendon. Theexpression of VGluT2 mRNA and protein wasupregulated in association with tendinosis, sug-gesting that locally derived glutamate may playroles in the pathology or in an attempted (i.e.,failed) repair response. Local regulation of gluta-mate levels may influence key tenocyte responsesto injury and/or mechanical loading, includingproliferation, apoptosis, and extracellular matrixmetabolism. Further studies of the role of gluta-mate signaling in tendon may shed light on theprocesses of mechanotransduction and overuseinjury.

ACKNOWLEDGMENTS

The authors would like to thank Ms. Ulla Hedlund forexcellent technical services. Funding was received fromthe Umea University Faculty of Medicine (HA, SF),Swedish National Centre for Research in Sports (HA,SF), and the Canadian-Scandinavian Foundation andCanadian Institutes of Health Research (AS).

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VGluT2 expression in painful Achilles and patellar tendinosis: Evidence of local glutamate release...

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