Targeting the NF- B signaling pathway in chronic tendon ...€¦ · Targeting the NF-kB signaling pathway in chronic tendon disease Adam C. Abraham1, Shivam A. Shah2, Mikhail Golman3,

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

TEND INOPATHY

1Department of Orthopedic Surgery, Columbia University, 650W 168th St, New York,NY 10032, USA. 2Department of Biomedical Engineering, Washington University inSt. Louis, 1 Brookings Drive, St. Louis, MO 63130, USA. 3Department of BiomedicalEngineering, Columbia University, 1210 Amsterdam Ave, New York, NY 10027, USA.4Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinaryand Life Sciences, University of Glasgow, 120 University Ave., Glasgow, ScotlandG12 8TA, UK. 5Departments of Orthopedic Surgery and Cell Biology and Physiology,Washington University in St. Louis, 660 S. Euclid Ave., St. Louis, MO 63110, USA. 6ShrinersHospital for Children, 4400 Clayton Ave, St. Louis, MO 63110, USA. 7Department of Or-thopedic Surgery, Mount Sinai, 5 E 98th St., New York, NY 10029, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected]

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Targeting the NF-kB signaling pathway in chronictendon diseaseAdam C. Abraham1*, Shivam A. Shah2*, Mikhail Golman3, Lee Song1, Xiaoning Li1, Iden Kurtaliaj3,Moeed Akbar4, Neal L. Millar4, Yousef Abu-Amer5,6, Leesa M. Galatz7, Stavros Thomopoulos1,3†

Tendon disorders represent the most common musculoskeletal complaint for which patients seek medical attention;inflammation drives tendondegeneration before tearing and impairs healing after repair. Clinical evidence has implicatedthe nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway as a correlate of pain-free return tofunctionafter surgical repair.However, it is currentlyunknownwhether this response is a reaction toor adriverofpathology.Therefore, we aimed to understand the clinically relevant involvement of the NF-kB pathway in tendinopathy, to determineitspotential causative roles in tendondegeneration, and to test its potential as a therapeutic candidate. Transcriptional profil-ingof early rotator cuff tendinopathy identified increases inNF-kBsignaling, including increasedexpressionof the regulatoryserine kinase subunit IKKb, which plays an essential role in inflammation. Using cre-mediated overexpression of IKKb intendon fibroblasts,weobserveddegenerationofmouse rotator cuff tendonsand theadjacenthumeral head. These changeswere associated with increases in proinflammatory cytokines and innate immune cells within the joint. Conversely, geneticdeletion of IKKb in tendon fibroblasts partially protected mice from chronic overuse–induced tendinopathy. Furthermore,conditional knockout of IKKb improved outcomes after surgical repair, whereas overexpression impaired tendon healing.Accordingly, targeting of the IKKb/NF-kB pathway in tendon stromal cells may offer previously unidentified therapeuticapproaches in the management of human tendon disorders.

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INTRODUCTIONTwenty percent of all consultationsmade to primary care physicians arerelated to musculoskeletal diseases; 30% of these are associated withtendon injuries, namely tendinopathies (1, 2), representing a highlyprevalent problem inmusculoskeletalmedicine. In the shoulder, rotatorcuff tendinopathy is a frequent source of pain and disability for morethan 17 million individuals in the United States alone, leading to lostdays from work, occupational challenges, recreational limitations, andincreased likelihood of tendon tears (3). Unfortunately, treatment out-comes are variable, operativemanagement is equivalent to nonoperativecare in some cases (4–6), and surgical repair failure rates range from 20to 94% (7–10). Factors associatedwith failure include tear size, chronicity,patient age, and other environmental factors (9, 11, 12). Therefore, dis-secting the cellular and molecular processes of tendon degenerationand healing after surgical repair will allow clinicians to implement pre-ventative interventions and prescribe therapeutics to improve outcomes.

The role of inflammation in tendon degeneration and healing hasbeen debated over the past three decades. Growing evidence supportsits fundamental role in disease progression (2, 13–15). It is hypothesizedthat chronic inflammation may drive degeneration before tearing andmay lead to fibrovascular scarring during healing and therefore is anattractive target for therapeutic intervention (15–17). Targeted blockadeof inflammatory mechanisms after tendon repair can improve pre-clinical outcomes, but the mechanism of action remains unknown(18–20). Recent clinical investigations have revealed that early stages

of tendinopathy involve nuclear factor kappa-light-chain-enhancerof activated B cells (NF-kB) (13, 15), a protein complex that controlscytokine production and apoptosis. Pathologic tendon stromal cellsexhibit phenotypic plasticity with higher NF-kB target gene expressionevenwithout proinflammatory stimulus (21). Dissecting the pivotal rolethat the NF-kB signaling pathway plays inmediating tendinopathy andtendon healing is crucial to developing therapeutic strategies.

The canonical NF-kB pathway constitutes a family of “rapid-acting”protein complexes that are bound to dimer-specific inhibitor of NF-kBproteins (IkB) andheld in latencywithin the cytoplasm(22). Inflammatorystimuli, acting through receptor-specific mechanisms, induce the re-cruitment of the IkB kinase (IKK) complex, which phosphorylatesIkB, leading to its degradation. NF-kB dimers are then free to trans-locate to the nucleus and induce transcription. Thus, IKK is an in-dispensable fulcrum for NF-kB signaling. The IKK complex iscomposed of the scaffolding protein NF-kB essential modifier(NEMO) and catalytic subunits IKKa and IKKb, the latter of whichregulates inflammation and fibrosis by targeting the IkBa:p50:p65NF-kB complex. Therefore, specifically restraining IKKb expressionor activity may slow or arrest tendinopathy progression andmay im-prove healing outcomes without altering other essential physiologicactivities of the NF-kB pathway. Here, we (i) confirm increased ca-nonical NF-kB signaling in human rotator cuff tendinopathy, (ii) re-capitulate tendon degeneration through persistent IKKb activationin tendon stromal cells in vivo, (iii) identify IKKb as a necessarycomponent of tendinopathy in animal models, and (iv) demonstratethe therapeutic potential of blocking IKKb activity.

RESULTSNF-kB signaling is increased in clinical tendinopathyClinical samples of early-stage tendinopathy exhibited dysregulation ofmore than 65% of the NF-kB–associated genes assayed (Fig. 1, A andB). Transcripts of the NF-kB complex subunits [Nfkb1: mean, 6.95-foldincrease (P = 0.019); Rel: mean, 8.41-fold increase (P = 0.007); Relb:

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mean, 13.53-fold increase (P=0.019); Fig. 1C] and IKKcomplex proteins[Chuk: mean, 11.47-fold increase (P < 0.001); Ikbkb: mean, 6.71-foldincrease (P = 0.045); Fig. 1D] were up-regulated in patients with rotatorcuff disease. Because NF-kB signaling is present in the pathogenesisof rotator cuff disease, and IKKb is essential for p65 phosphorylation(23) and nuclear translocation (Fig. 2A), we next asked whetherIKKb activation in tendon stromal cells was sufficient to drive tendondegeneration.


Chronic IKKb overexpression inmurine tendon fibroblasts mimicshuman rotator cuff diseaseConditional modulation of IKKb wasachieved using the tendon-specificscleraxis (Scx) promoter (24) drivingcre-mediated recombination of LoxPsites flanking either exon 7 of the Ikbkbgene (IKKbKOScx) or a stop codon pre-ceding a constitutively active form ofIKKb within an inserted Rosa26 locus(IKKbCAScx) (Fig. 2B) (25,26). IKKbCAScx

mice exhibited noticeable hair loss by8 weeks old, which became progressivelyworse throughout adulthood, whereasIKKbKOScx mice had no overt pheno-type (Fig. 2C). Cultured tendon fibro-blasts from IKKbCAScx mice respondedto interleukin-1b (IL-1b) stimulus withincreased expression of the downstreamtranscriptional targets tumor necrosisfactor–a (Tnfa) and prostaglandin-endoperoxide synthase 2 (Ptgs2) of theNF-kB pathway in a dose-dependentmanner (Tnfa, P = 0.0024; Ptgs2 ,P < 0.0001; fig. S1). Expression ofmatrixmetalloproteinase (MMP)–1a, MMP-3,andMMP-13was significantly decreasedin IKKbKOScx compared to wild-type(WT) and IKKbCAScx tendon fibroblastsin vitro (MMP-1a: P < 0.0001, MMP-3:P < 0.0001, and MMP-13: P < 0.0001;fig. S1). Expression of Scx, a marker oftendon fibroblast differentiation, was notaffected by IKKb modulation, whereasexpression of tenomodulin (Tnmd) wassignificantly increased in IKKbCAScx

compared to WT and IKKbKOScx ten-don fibroblasts (Tnmd,P



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Fig. 2. Modulation of IKKb expression in murine tendon fibroblasts. (A) Schematic of NF-kB signaling and gene transcription. NF-kB signaling was controlled by targetinginhibitor of NF-kB kinase subunit b (IKKb), which acts upstream of the NF-kB complex. Tendon fibroblast IKKb modulation was achieved by deletion of IKKb (IKKbKOScx) andactivation of IKKb (IKKbCAScx) using Cre-loxP–mediated recombination under the Scx promoter. (B) Expression of IKKb in tendon fibroblasts from WT, IKKbKOScx, and IKKbCAScx

mice. Culturedmouse osteoclasts were used as a positive control (pos. CTL) (51). (C) Photograph of 16-week-oldmice to demonstrate hair loss. (D) Secreted cytokines and growthfactors in vehicle and IL-1b–treated tendon fibroblasts from WT, IKKbKOScx, and IKKbCAScx mice (n = 5 per group). (E) Immunolabeling for CD68 (brown) in the supraspinatustendon fromWT, IKKbKOScx, and IKKbCAScx mice. T, tendon; E, enthesis. (F) Microcomputed tomography (mCT) three-dimensional reconstruction of coronal section from proximalhumerus. Arrows denote the supraspinatus tendon attachment site. (G) Quantification of bone morphometry: Bone volume normalized to total volume (BV/TV), trabecularthickness (Tb.Th), cortical thickness (Ct.Th), and total cortical area (Tt.Ar) (n = 8 to 9 per genotype). (H) Quantification of mechanical properties of the supraspinatus tendon-to-bone attachment (n = 8 to 9 per genotype). Data are shown as means ± SD with individual points representing biologically independent samples. Statistically significantdifferences were calculated using one-way analysis of variance (ANOVA) (genotype) with Fisher’s least significant difference (LSD) post hoc test. ****P < 0.0001, ***P < 0.001,**P < 0.01, *P < 0.05.

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tendon and epitenon) and loss of metachromasia at the enthesis(implying a loss of proteoglycans at the fibrocartilaginous tendon-to-bone attachment) (Fig. 2E, fig. S3, and table S1). Furthermore,IKKbCAScx mice exhibited structural and functional losses of therotator cuff, including less cortical and trabecular bone of the humeralhead (Fig. 2G) and reduced tendon mechanical properties (Fig. 2H).These outcomes are similar to patients with rotator cuff disease, whoalso present with increased CD68+ tissue-resident macrophages(13, 15), loss of cortical and cancellous bone near the tendon enthesis(29), and increased compliance of the tendons (30). On the basis ofthese data, we next investigated the therapeutic potential of modulatingthe IKKb/NF-kB signaling pathway for mitigating tendinopathy.

Chronic overuse degrades tissue functionDownhill treadmill running is a rodent model of overuse tendinopathy(31, 32); however, how closely this model aligns with clinical cases ofrotator cuff disease and NF-kB signaling is unknown. Four weeks ofoveruse did not induce chronic dysregulation of NF-kB signaling(Fig. 3, A and B). Histologically, the tendons and tendon enthesesof IKKbKOScx mice appeared similar to those in WT mice (Fig. 3C).The tendons were populated with spindle-shaped fibroblasts alignedwith the direction of collagen fibers. The tendon entheses were pop-ulated by chondrocytes aligned in columns from tendon to bone. Therewere increased cell numbers and a loss of cell organization in thetendons and tendon entheses of IKKbCAScx mice.

Treadmill running down-regulated Ikbkb and tendon-associated(Scx, Col1a1, Col3a1, and Bgn) transcripts (Fig. 3D) across genotypeswhen compared to cage activity controls.WTmice exhibited degenera-tion of the tendon-to-bone attachment mechanical properties due tooveruse (Fig. 3E). Abolishing IKKb signaling in IKKbKOScx mice pro-tected from losses of ultimate stress and Young’smodulus (Fig. 3E). Ac-tivation of IKKb in IKKbCAScx mice, coupled with treadmill running,did not worsen the mechanical properties compared to cage activity.Overuse did not have an apparent effect on the bone microstructure(fig. S4). These results suggest that changes in tendon function due tochronic overuse are not solely dependent on the IKKb/NF-kB signalingaxis. To examine the role ofNF-kB signaling after a tendon tear, we nextinvestigated murine acute rotator cuff injury with immediate repair.

Blocking IKKb improves surgical repair outcomes afteracute injuryTo examine the role of IKKb during tendon healing, mice underwentsurgical transection and immediate repair of the supraspinatus tendon(Fig. 4, A and B). Injury and repair led to increased NF-kB signalingwithin the tendon 2 weeks after surgery (Fig. 4C), including up-regulation of protein coding genes for the IKK andNF-kB complexes(Fig. 4D). Injury and repair resulted in significant hypertrophy of thesupraspinatus tendons fromWTand IKKbKOScxmice (WT:P=0.0051and IKKbKOScx: P = 0.0024; Fig. 4E). The structural andmaterial prop-erties of the tendon-to-bone attachment were reduced inWTmice. De-letion of IKKb resulted in no significant differences in stiffness (P = 0.21),resilience (P = 0.29), or Young’s modulus (P = 0.13) when compared tocontralateral sham operation limbs (Fig. 4E). Constitutive activation ofIKKb suppressed healing, resulting in significantly lower tendon failurestrength (P = 0.033). The acute injury and repair procedure did not haveany apparent effect on the bone microstructure (fig. S5). On the basis ofthese results, we tested the potential therapeutic efficacy of pharmaco-logical inhibition of IKKb using a small-molecule inhibitor in an in vitrohuman inflammatory tendinopathy model.


In vitro inhibition of IKKb in human tendon stromal cellsblocks NF-kB signaling and cytokine productionTendon fibroblasts were isolated from healthy human hamstringtendons and cultured with IL-1b to model inflammatory tendinopathyin vitro. IKKb inhibitor VIII successfully repressed transcription ofmost NF-kB signaling genes in the IL-1b–treated fibroblasts (Fig. 5A).Production of IL-6 and CCL-2 was reduced to control concentrationsby the inhibitor treatment (Fig. 5B). These two cytokines were themost abundant in cultured tendon fibroblasts from IKKbCAScx miceand are present in human tendinopathy (17). Treatment did not signif-icantly change the expression of extracellular matrix transcripts Col1a1or Col3a1 (Col1a1: P = 0.21 and Col3a1: P = 0.61; fig. S6). Together,pharmacological inhibition of IKKb desensitized human tendon fibro-blasts to IL-1b stimulation in vitro while maintaining their transcrip-tional identity.

DISCUSSIONThe NF-kB signaling pathway plays a central role in inflammation,stress response, and cell survival (22). In this study, we observed in-creases in NF-kB signaling in the subscapularis tendons of patients un-dergoing surgical repair of the rotator cuff. Intact subscapularis tendonsin rotator cuff disease served as a model for the early stages of tendino-pathy due to mechanical overuse (33). Studies of early-stage tendino-pathic tissues, including the subscapularis, have previously revealedincreases in NF-kB complex protein expression, downstream proin-flammatory cytokine transcripts, and immune cell recruitment and ac-tivation (13, 15). This inflammatory phenotype persists in patients withchronic pain after subacromial decompression treatment (17, 21). Spe-cifically, fibroblasts within the diseased tendon microenvironmentcontinued to exhibit phenotypic “memory” 4 years after treatment, wereprimed to bemore responsive to IL-1b stimulation in vitro, and did notresolve mRNA expression of IL-6 and IL-8 after cessation of stimulus(21). Dakin et al. (21) proposed that this memory function is a crucialmechanism in the development of chronic inflammation. Therefore,resolution of NF-kB–mediated inflammation may be critical for suc-cessful outcomes in human tendon disease.

NF-kB may drive degeneration before tearing and impair healingafter surgical repair. To better understand the intrinsic degenerativemechanisms of rotator cuff tendinopathy, NF-kB was selectively ac-tivated or inhibited in tendon fibroblasts of geneticallymodifiedmice(fig. S7). Analysis of mice with constitutively active IKKb demonstratedthat activation of the canonical NF-kB pathway was sufficient to inducedegeneration in vivo, paralleling hallmarks of rotator cuff disease.Tendon stromal cells in these mice perpetually secreted various cy-tokines that are present in exercise adaptation and clinical tendinopathy(27, 34, 35). Cytokines of the interleukin family can exhibit variouseffects on tendon extracellular matrix remodeling. For example, IL-6increases total collagen synthesis (36); however, IL-17 and IL-33 in-crease the ratio of type III to type I collagen and up-regulate MMP pro-duction (17). In addition to interleukins, we found that the tendonstromal compartment from IKKbCAScx mice produced many chemo-tactic (CCL-2, CCL-5, CXCL1, CXCL2, and CXCL10) and G-CSFproteins with and without IL-1b stimulation. These factors may drivethe observed increases in CD68+ cells in tendons from IKKbCAScx

mice. Clinical samples of early- and advanced-stage disease tendonsexhibit active recruitment of CD14+ monocytes that mature intoCD68+ macrophages (13, 15). Changes in tendinopathy gene and pro-tein signatures are believed to follow macrophage activation. However,

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Fig. 3. Modulation of IKKb/NF-kB signaling with chronic overuse. (A) Ten-week-old mice were subjected to a chronic overuse protocol with 1 week of progressive training,followed by 4 weeks of downhill running. Control mice were permitted normal cage activity. (B) NF-kB pathway–related gene regulation due to overuse. (C) Hematoxylin andeosin (H&E) images of WT, IKKbKOScx, and IKKbCAScx mice. B, bone; black arrowhead, spindle-shaped tendon fibroblast; white arrowhead, enthesis chondrocyte. (D) mRNAexpression of Ikbkb, IL-1b, Scx, Col1a1, Col3a1, and Bgn in tendon from control cage-active or treadmill overuse–subjected WT (n = 4 to 6 per group), IKKbKOScx (n = 3 to 4per group), and IKKbCAScx (n = 3 per group) mice. (E) Failure load, ultimate stress, and Young’s modulus of the supraspinatus tendon-to-bone attachment in cage-active andtreadmill overuse–subjected mice (n = 5 to 13 per group). Data are shown as means ± SD with individual points representing biologically independent samples. Statisticallysignificant differences were calculated using two-way ANOVA (genotype, overuse) with Fisher’s LSD post hoc test. **P < 0.01 and *P < 0.05.

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recent studies of fibroblast-macrophage signaling circuits in vitro de-monstrated that dysregulation of the stromal compartment results inuntetheredmacrophage expansion and activation, potentially overload-ing organ-carrying capacity and leading to degeneration (37). After atendon tears, animal models have shown that macrophages are theprimary immune cell at the site of injury and promote scar formation,cell death, and matrix degradation (38). Modulating macrophage be-havior by promoting M2 polarization using adipose-derived stem cells


and growth factors has a protective effect on tendon fibroblasts in thiscontext (39, 40). Yet, tendon healing may also be improved by physicalactivity that drives a robust inflammatory response characterized by anincreased cytokine transcription and a delayed switch to M2 polariza-tion (41, 42). However, detensioning of engineered human tendontissue also increases the expression of proinflammatory mediatorsand alters the response to growth factors, indicating that properlybalanced mechanical loading is required to develop, maintain, and heal

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Fig. 4. Modulation of IKKb/NF-kB signaling with acute supraspinatus injury and repair. (A) Experimental protocol. Ten-week-old mice were subjected to a unilateral acuteinjury of the supraspinatus tendon and immediate repair, followedby 2weeks of recovery. Shamoperationswere performedon contralateral limbs. (B) H&E image of the repairedtendon and newbone formation around the suture tunnel after 2weeks of recovery. GP, growth plate; S, suture hole; AC, articular cartilage. The mCT image (right) shows the bonetunnel (BT) below the epiphysis. (C) NF-kB signaling gene expression 2 weeks after acute injury and repair (n = 4 to 5 per group). (D) mRNA expression of IKK complex–relatedgenes and NF-kB complex–related genes in tendon 2 weeks after recovery (n = 4 to 5 per group). (E) Quantification of murine tendon cross-sectional area (CSA), stiffness, failureload, resilience, Young’s modulus, and ultimate stress 2 weeks after injury and repair (n = 4 to 5 per group). Data are shown as means ± SD with individual points representingbiologically independent samples. Statistically significant differences were calculated using two-way ANOVA (genotype, injury) with Fisher’s LSD post hoc test. ***P < 0.01,**P < 0.01, *P < 0.05.

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tendons (43). Thus, further studies examining the cross-talk amongresident stromal, immune-sensing, and infiltrating compartments, aswell as mechanical loading in tendinopathy and healing, are required.

At the tissue level, the constitutive activation of IKKb resulted inmechanically weaker attachment of the supraspinatus tendon to thehumeral head compared to WT controls. Recent advances in ultra-sound elastography have revealed that clinically diagnosed tendinopathycorrelates with increased compliance of the tendon (44). Similar resultshave been observed in many animal injury models (38), including thetreadmill overuse protocol. We also found a loss of bone microstructurewithin the adjacent humeral head. Similarly, patients with partial- andfull-thickness rotator cuff tears lose bone mass near the tendon insertionsite, which might be attributed to decreased mechanical loading after atear (29). However, we found that the constitutive activation of thecanonical NF-kB pathway in tendons can also drive bone loss, suggest-ing a cellular signaling network beyond the local microenvironment.

To determine the role of the canonical NF-kB pathway in clinicallyrelevant pathologic scenarios, we genetically ablated IKKb-mediatedsignaling in the tendon fibroblasts of mice and subjected them totreadmill overuse (tomodel tendinopathy before a tendon tear) or acute


supraspinatus tendon injury and repair(to model tendon healing after surgicalrepair). In both cases, IKKbKOScx micewere partially protected from biomech-anical degeneration compared to WTcontrols or IKKbCAScx mice. In vitro,IKKbKOScx tendon fibroblasts wereprotected from IL-1b stimulus: Theyshowed decreases in transcription ofmatrix turnover enzymes (Mmp-1a,Mmp-3, and Mmp-13) and in transla-tion of proinflammatory cytokines andchemokines, while maintaining tenogenicmarkers (Scx) and matrix production(Col1a1). These data support the idea thatNF-kB signaling is a key checkpoint forsustaining inflammatory signaling net-works and directing tendon remodeling(17, 27). Many downstream effectors,such as cytokines, have been found atthe tissue level in human and animalmodels of tendinopathy; however, thereis limited evidence for increases in NF-kB signaling specifically in nonmyeloidcells of diseased tendons (14, 15, 21).Our findingsmechanistically demonstratethe capacity of the tendon stromal com-partment to shape the healing niche andprovide a molecular target for improvingthe treatment of clinical tendinopathy. Apotential crux of NF-kB–directed therapyis specifically targeting tendon stromalcells while allowing resident and infiltrat-ing immune cells to initiate tissue repair.Leveraging technology such as nanoparticle-based delivery of NF-kB–targeted smallinterfering RNA in a cell-specific mannercould provide a platform for advancingtendon therapeutics (45).

A limitation of the human biopsy portion of the study was the useof hamstrings tendon as healthy control samples. A second limitation isthat murine models of tendinopathy cannot fully recreate the humancondition (46). We chose to examine two different models of clinicallyrelevant tendinopathy that address various aspects of tendon remodelingand surgically repaired healing. Although we did not observe the chronicdysregulation of NF-kB signaling due to treadmill overuse, IKKbKOScx

mice were partly protected from degeneration. These results highlight apossible novel role of this pathway in tendon fibroblasts. Last, the in vitromodel demonstrated therapeutic potential for suppressing the NF-kBpathway but only provides a limited model of inflammatory tendinopa-thy (i.e., inflammationwasmodeledwith a single cytokine). Future in vivostudies are necessary to determine the efficacy of IKKb inhibition fortreating tendinopathy and/or improving tendon healing.

In summary, we observed increases in the canonical NF-kB sig-naling in patients with rotator cuff tendinopathy. Overexpression ofIKKb inmurine tendon fibroblasts recreated rotator cuff disease in vivo.Inhibiting IKKb in tendon fibroblasts limited biomechanical degenera-tion of the supraspinatus tendon due to overuse injury and partially im-proved healing after surgical repair.

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f NF-kB signaling array in healthy human tendonbroblasts treated with IL-1b with or without IKKbhibitor. (B) Proinflammatory cytokines IL-6 andCL-2 produced by healthy human tendon fibroblastseated with IL-1b with or without IKKb inhibitor. Datare shown as means ± SD. Statistically significant dif-rences were calculated using one-way ANOVA (treat-ent) with Fisher’s LSD post hoc test. *P < 0.05.

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MATERIALS AND METHODSStudy designThe goal of this study was to investigate the role of the NF-kB pathwayin rotator cuff degeneration and healing.We compared gene signaturesbetween patients with early-stage rotator cuff tendinopathy and controlhamstrings samples from patients undergoing anterior cruciate liga-ment reconstruction. We further explored the mechanistic role of thissignaling pathway in vivo using genetically modified mice by intro-ducing gain- and loss-of-function IKKb mutations in tendon fibro-blasts. Last, we hypothesized that ablating IKKb/NF-kB signaling couldprotect tendon fibroblasts from inflammation in the context of chronicoveruse and acute injury and repair. Statistical analysis and sample sizeswere determined from previous studies (13, 16, 39, 47) that were suf-ficiently powered to detect meaningful differences in clinical tendino-pathy samples, murine rotator cuff microstructure, and biomechanicalfunction and using an in vitro model of tendinopathy. Samples for bio-mechanical testing, mCT, and histological scoring were randomized,and analysis was performed by a blinded investigator. Sample sizeand replication are provided in the figure legends.

Study approvalHuman study procedures and protocols were approved by the NationalHealth Service West of Scotland Ethics Committee (REC 11/S0704/7).Full informed consent was obtained from all patients. Animal studieswere approved by Washington University and Columbia UniversityInstitutional Animal Care and Use Committees.

Clinical samplesSamples of “early-stage” tendinopathy were obtained using a previouslyestablished protocol (13). The subscapularis tendon (n = 5; age, 33 to56 years) was obtained from patients with rotator cuff tears undergoingreparative surgery using a standard three-portal technique. The tendonwas arthroscopically biopsied from the superior border of the tendon1 cm lateral to the glenoid labrum. Control hamstring tendons (con-firmed by H&E staining; Bonar score 1) were taken from patients un-dergoing anterior cruciate ligament autograft and used as healthycontrols for gene expression (n = 4; age, 22 to 44 years) or primary cellextraction (n = 3).

Generation of tendon-specific IKKb miceGeneticmanipulation of the canonicalNF-kB signaling in tendon fibro-blasts was achieved using cre-loxP cross-breeding. Tendon specificitywas determined using an Scx promoter driving a cre-recombinase(ScxCre) sequence in a bacterial artificial chromosome transgene (48).IKKb expression was ablated in tendon fibroblasts by crossing ScxCremice with Ikbkbtm2Cgn (IKKb-floxed) mice to yield IKKbKOScx (25).IKKb overexpression was accomplished using Gt(Rosa26)tm4(Ikbkb)Rsky

(IKKbCA-floxed) mice harboring an internal ribosomal entry site–enhanced green fluorescent protein and complementary DNA (cDNA)sequence encoding Ikbkb preceded by a floxed stop codon to yieldIKKbCAScx (26). All initial breeding pairs were set up with 8-week-oldmice to produce the 31 female and 83malemice used in this study [WT,n = 57 (19 females and 38 males); IKKbKOScx, n = 29 (6 females and23 males); and IKKbCAScx, n = 28 (6 females and 22 males)].

Tendon fibroblast isolation and cultureAfter euthanasia, tail tendons were dissected from mice, minced,and placed in alpha-modified Eagle’s medium (alpha-MEM; Gibco).Tissue was digested in 0.2% collagenase type A (Sigma-Aldrich) in


phosphate-buffered saline for 3 hours. The digested tissue was passedthrough a 100-mm cell strainer, pelleted by centrifugation at 460g for5 min; the supernatant was discarded; and the cells were resuspendedand plated in supplemented culture medium [alpha-MEM with 10%fetal bovine serum(Sigma-Aldrich), 1%penicillin/streptomycin (Gibco),and 1% amphotericin B (Invitrogen)] at 37°C, 5% CO2, and 95%humidity. The cells were used on passage 2. Human tendon–derivedcells were extracted from biopsied hamstring tendon explants (age, 22to 44 years). Cultures were maintained at 37°C in a humidified atmo-sphere of 5%CO2 for 28 days. Cells were subcultured and trypsinized atsubconfluency and used at passage 3.

In vitro model of tendinopathyMouse tendon stromal cells were plated in six-well plates at a density of2 × 106 cells perwell with 1.5ml of supplemented culturemedium.After24 hours, cells were treated with IL-1b (10 ng/ml; R&D Systems). Theculture medium was collected after 24, 48, and 72 hours and stored at−80°C. Human tendon cells were plated in 12-well culture plates at adensity of 5 × 104 cells per well with 1 ml of supplemented culture me-dium, expanded for 48 hours, treated with 1 ng of IL-1b, 50 mM IKKbinhibitor VIII (MilliporeSigma), or both for 4 hours, and compared tovehicle-treated controls. The supernatant was sampled to determineinflammatory cytokine concentration, and RNA was isolated fromcell lysates.

Treadmill overuse modelChronic overuse tendinopathy was established by subjecting10-week-old mice (WT, n = 8 females and n = 22 males; IKKbKOScx,n = 3 females and n = 12 males; and IKKbCAScx, n = 2 females andn = 11 males) to 4 weeks of treadmill running at speeds of 20 m/minfor 30 min/day, five times a week at a decline of 10°. Before protocolinitiation, mice were subjected to a training week consisting of 5 minof running on the first day, followed by increasing durations by5 min/day until 25 min of running was achieved on the fifth day.Cage activity mice served as controls (WT, n = 16; IKKbKOScx,n = 8; and IKKbCAScx, n = 8).

Acute injury and repair modelShoulder injury and surgical repairmethods were adapted fromBell et al.(49). Ten-week-old mice (WT, n = 7 females and n = 6 males;IKKbKOScx, n = 3 females and n = 4 males; and IKKbCAScx, n = 2females and n = 5 males) were anesthetized using isoflurane andplaced in a left lateral decubitus position. An incision in the skinwas made to expose the deltoid. The deltoid was released to visualizethe humerus, which was grasped with microforceps for stability. An 8-0Ethilon suture was used to place a modified Mason-Allen stitch in thesupraspinatus tendon. After the tendon grasping suture, the supraspi-natus tendon was sharply detached from the humeral head. A 27-gaugeneedle was used to create a bone tunnel in the humeral head below thegrowth plate. The suture was then ligated through the bone tunnel, re-pairing the supraspinatus tendon to its original attachment site. The del-toid was reflected back over the humerus, and the skinwas closedwith a5-0 PROLENE suture. After acute injury and repair, mice were allowedfree cage activity and euthanized after 2 weeks. The uninjured contra-lateral shoulder was used as uninjured control.

Gene expressionTissues were snap-frozen in liquid nitrogen and physically disruptedusing a ball mill homogenizer (Mikro-Dismembrator U, Sartorius).

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RNA extraction was performed using guanidinium thiocyanate–phenol–chloroform (TRIzol, Thermo Fisher Scientific) and interphaseseparation (Phase Lock Gel, QuantaBio). RNA cleanup was performedusing spin columns (RNeasy Mini Kit, Qiagen) with on-columndeoxyribonuclease I treatment (Qiagen). Cells were lysed in lysis buffer(RLT Buffer, Qiagen), and RNA isolation was performed using spincolumns (RNeasy Mini Kit, Qiagen). RNA quantity and quality weredetermined using a spectrophotometer (NanoDrop 1000, ThermoFisher Scientific). For mouse studies, RNA was reverse-transcribedinto cDNA using the SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific). Quantitative real-time reverse transcription poly-merase chain reaction (qRT-PCR) was performed with TaqMan primers(Invitrogen) using a BioMark HD System. Gene expression changeswere measured for Ikbkb (IKKb, Mm01222247_m1), RelA (p65,Mm00501346_m1), RelB (p50, Mm01268877_m1), Chuk (IKKa,Mm00432529_m1), Ikbkg (NEMO, Mm00494927_m1), and Gapdh(Mm99999915_g1). NF-kB pathway profiling was performed usingspecies-specific prepared RT2 Profiler arrays (Qiagen). RNA wasreverse-transcribed into cDNAusing theHigh-Capacity cDNAReverseTranscriptionKit (Invitrogen). qRT-PCRwas performedusing PowerUpSYBR green (Invitrogen) and QuantStudio 6 Flex (Applied Biosystems).All gene expression data were analyzed using the DDCt method, withthe results first normalized to a housekeeping gene, as indicated in thefigures, and then again to WT expression.

Western blotCells were plated in 60-mm Petri dishes at a density of 3 × 106 in3 ml of supplemented culture medium. After 24 hours, supplementedculture medium was replaced with 2 ml of alpha-MEM for 2 hours.Cells were lysed with radioimmunoprecipitation assay lysis buffer(Thermo Fisher Scientific) supplemented with protease inhibitors(cOmplete, Sigma-Aldrich) and phosphatase inhibitors (PhosSTOP,Sigma-Aldrich). Total proteinwas quantifiedwith the Pierce BCAProteinAssay Kit (Thermo Fisher Scientific). Equal amounts of total cell lysate(100 mg) were subjected to SDS-electrophoresis on 8% acrylamide gels.Gels were run at a constant voltage of 100 mV (PowerPac 3000, Bio-Rad) and transferred onto a nitrocellulosemembrane with the Trans-BlotTurbo Transfer System (Bio-Rad). Immunoblotting was completed forIKKb (Cell Signaling Technology) and b-actin (Cell Signaling Tech-nology). A Li-Cor Odyssey scanner was used to visualize immunoblots,and ImageJ software (NIH) was used for quantification.

HistologyMouse shoulders were dissected, fixed in 4% paraformaldehyde, de-calcified in 0.5 M EDTA, and embedded in paraffin using standardtechniques. Sections (5 mm) were obtained and stained with H&E.

ImmunohistochemistryHistological sections were deparaffinized in xylene and rehydratedthrough graded ethanol. Endogenous peroxidase activity was quenchedusing 0.5% H2O2. Antigen retrieval was performed using Uni-Trieve(Innovex Biosciences). Blocking of nonspecific binding was performedusing 2.5% horse blocking serum (Vector Laboratories). Sections wereincubated overnight at 4°C with anti-CD68 (KP1, ab955, Abcam) orisotype control antibody. Staining of antigens was performed usingthe ImmPRESS Polymer Detection Kit (Vector Laboratories). Sectionswere counterstained using Gill’s hematoxylin (Vector Laboratories),differentiated in 2% glacial acetic acid, blued in 30% NH4OH, de-hydrated, andmounted using Cytoseal XYL (Thermo Fisher Scientific).


Cytokine/chemokine quantificationCytokine concentration from culture supernatant was determinedusing either single-antibody enzyme-linked immunosorbent assay(ELISA) kits (Invitrogen) or Milliplex Mouse Cytokine/ChemokineMagnetic Bead Panel (EMD Millipore) following the manufacturer’sprotocol. Expression of cytokines and chemokines with three bio-logical replicates and detectable concentrations was included foranalysis.

Microcomputed tomographyThe humerus with the supraspinatus tendon attached was dissected forbone morphometry analysis. Samples were scanned at an energy of55 kilovolt peaks (kVp), an intensity of 145 mA, and a standard resolu-tion of 12.3 mm(mCT 40, Scanco). Reconstructed images were evaluatedusing a segmentation algorithm to separate cortical and trabecular boneof the humeral head proximal to the growth plate (CTAn, Bruker).

Tendon-to-bone biomechanicsAfter mCT, the supraspinatus muscle was removed from the tendon inpreparation for biomechanics (50). The humerus was potted in epoxy(Parbond 101, McMaster-Carr), and samples were tested in a salinebath at 37°C (30 N load cell; ElectroPuls 1000, Instron Corp.). Uniaxialload-to-failure tensile tests consisted of five cycles of preconditioning(5% strain and 0.2%/s), 300-s rest, and then extension to failure at0.2%/s. Structural properties were determined from load-deformationdata. Material properties were determined from normalized load de-formation using tendon cross-sectional area measured from mCTand strain determined as grip-to-grip displacement relative to initialgauge length.

Statistical analysisAll data are shown as means ± SD. All statistical analyses, includingShapiro-Wilk normality test, ANOVA, Fisher’s least significantdifference (LSD) with Bonferonni correction for multiple comparisons,and Student’s t test, as indicated in the figure legends, were performedusing GraphPad Prism 7 software. A P value of



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Funding: This study was funded by the NIH (R01AR055580 and R01AR057836 to S.T.;5F31AR066452-03 to S.A.S.; and R01AR049192, R01AR054326, and R01AR072623, to Y.A.-A.),Shriners Hospitals for Children (Biomedical grant 86200 to Y.A.-A.), Medical ResearchCouncil, UK (MR/R020515/1 to M.A. and N.L.M.), and Arthritis Research UK (21346 to N.L.M.)Author contributions: Conception: A.C.A., S.A.S., and S.T.; research design: A.C.A., S.A.S.,


N.L.M., Y.A.-A., and S.T.; data acquisition/analysis: A.C.A., S.A.S., M.G., L.S., X.L., I.K., M.A.,N.L.M., Y.A.-A., L.M.G., and S.T.; resource assistance: M.A., N.L.M., Y.A.-A., and L.M.G.; writing,drafting, and editing: A.C.A., S.A.S., and S.T.; project administration: S.T. Competinginterests: The authors declare that they have no competing interests. Data and materialsavailability: All data associated with this study are present in the paper or in theSupplementary Materials.

Submitted 14 September 2018Accepted 31 January 2019Published 27 February 201910.1126/scitranslmed.aav4319

Citation: A. C. Abraham, S. A. Shah, M. Golman, L. Song, X. Li, I. Kurtaliaj, M. Akbar, N. L. Millar,Y. Abu-Amer, L. M. Galatz, S. Thomopoulos, Targeting the NF-kB signaling pathway in chronictendon disease. Sci. Transl. Med. 11, eaav4319 (2019).

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B signaling pathway in chronic tendon diseaseκTargeting the NF-

Millar, Yousef Abu-Amer, Leesa M. Galatz and Stavros ThomopoulosAdam C. Abraham, Shivam A. Shah, Mikhail Golman, Lee Song, Xiaoning Li, Iden Kurtaliaj, Moeed Akbar, Neal L.

DOI: 10.1126/scitranslmed.aav4319, eaav4319.11Sci Transl Med

.βin blocking IKKB target gene transcription. These results suggest that there may be therapeutic potentialκshowed repressed NF-

inhibitorβa surgical model of tendon injury and repair, and human tendon stromal cells treated with an IKKinduced overuse tendinopathy model and in−mice prevented maladaptive tendon remodeling in a treadmill running

in tendon in β. Genetic deletion of IKKβwhich was mimicked in mouse tendon fibroblasts overexpressing IKKB,κinhibiting the pathway. Clinical tendon samples of human rotator cuff disease showed up-regulation of NF-

investigated how this signaling pathway causes tendinopathy and potential therapeutic effects ofet al.Abraham B signaling contribute to tendon degeneration and injury, such as rotator cuff injury.κInflammation and NF-

Targeting tendinopathy

ARTICLE TOOLS http://stm.sciencemag.org/content/11/481/eaav4319

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2019/02/25/11.481.eaav4319.DC1

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Targeting the NF- B signaling pathway in chronic tendon ...€¦ · Targeting the NF-kB signaling pathway in chronic tendon disease Adam C. Abraham1*, Shivam A. Shah2*, Mikhail Golman3,

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Targeting the NF- B signaling pathway in chronic tendon ...€¦ · Targeting the NF-kB signaling pathway in chronic tendon disease Adam C. Abraham1, Shivam A. Shah2, Mikhail Golman3,