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Neuropeptide and catecholamine effects on tenocytes in tendinosis development: studies on two model systems with focus on proliferation and apoptosis
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Achilles tendinopathy is a common clinical syndrome of chronic Achilles tendon pain combined with thickening of the tendon and impaired tendon function. Tendinopathy is often, but not always, induced by mechanical overload, and is frequently accompanied by abnormalities at the tissue level, such as hypercellularity and angiogenesis, in which case the condition is called tendinosis. In tendinosis, there are no signs of intratendinous inflammation, but occasionally increased apoptosis is observed. Tendinosis is often hard to treat and its pathogenesis is still not clear. Recently, a new hypothesis has gained support, suggesting a biochemical model based on the presence of a non-neuronal production of classically neuronal signal substances by the primary tendon cells (tenocytes) in tendinosis. The possible functional importance of these signal substances in tendons is unknown and needs to be studied. In particular, the neuropeptide substance P (SP) and catecholamines are of interest in this regard, since these substances have been found to be up-regulated in tendinosis. As both SP and catecholamines are known to exert effects in other tissues resulting in changes similar to those characteristic of tendinosis, it is possible that they have a role in tendinosis development. It is furthermore unknown what elicits the increased intratendinous neuropeptide production in tendinosis, but given that tendon overload is a prominent riskfactor, it is possible that mechanical stimuli are involved.

The hypothesis of this thesis work was that intratendinous production of SP is up-regulated in response to load of Achilles tendons/tenocytes, and thatstimulation of the preferred SP receptor, the neurokinin-1 receptor (NK-1 R), aswell as stimulation of the catecholamine α2 adrenoreceptors, contribute to the hypercellularity seen in tendinosis, via increased proliferation and/or decreased apoptosis, and that SP stimulates tendon angiogenesis. The purpose of the studies was to test this hypothesis. To achieve this, two model systems were used: One in vivo (rabbit Achilles tendon overload model of tendinosis) and one in vitro (human primary Achilles tendon cell culture model).

Results: In the rabbit Achilles tendon tissue, SP and NK-1 R expression was extensive in the blood vessel walls, but also to some extent seen in the tenocytes. Quantification of endogenously produced SP in vivo confirmed intratendinous production of the peptide. The production of SP by human tendon cells in vitro was furthermore demonstrated. The catecholamine synthesizing enzyme tyrosine hydroxylase (TH), as well as the α2A adrenoreceptor (α2A AR), were detected in the tenocytes, both in vivo in the rabbit tissue and in vitro in the human tendon cells. As a response to mechanical loading in the in vivo model, the intratendinous levels of SP increased, and this elevation was found to precede distinct tendinosis changes. The in vitro model demonstrated the same response to load, i.e. an increased SP expression, but in this case also a decrease in the NK-1 R expression. In the in vivo model, exogenously administered SP, as well as clonidine (an α2 AR agonist), accelerated tenocyte hypercellularity, an effect that was not seen when administrating a specific α2A AR antagonist. Exogenous administration of SP also resulted in intratendinous angiogenesis and paratendinous inflammation. In the in vitro model, both SP and clonidine had proliferative effects on the human tenocytes, specifically mediated via NK-1R and α2A AR, respectively; both of which in turn involved activation/phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Exogenously administered SP, in Anti-Fas induced apoptosis of the tenocytes in vitro, confirmed SP to have an anti-apoptotic effect on these cells. This effect was specifically mediated via NK-1 R and the known anti-apoptotic Akt pathway.

Conclusions: In summary, this thesis concludes that stimulation of NK-1 R and α2A AR on tenocytes, both in vitro and in vivo, mediates significant cell signalling effects leading to processes known to occur in tendinosis, including hypercellularity. The pathological role of the hypercellularity in tendinosis is still unclear, but it is likely to affect collagen metabolism/turnover and arrangement, and thereby indirectly tendon biomechanical function. Additional evidence is here provided showing that SP not only causes tenocyte proliferation, but also contributes to anti-apoptotic events. Furthermore, it was concluded that SP may be involved in the development of tendinosis, since its production is increased in response to load, preceding tendinosis, and since SP accelerates tendinosis changes, through some mechanistic pathways here delineated. These findings suggest that inhibition of SP, and possibly also catecholamines, could be beneficial in the reconstitution/normalization of tendon structure in tendinosis.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2013. , 80 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1572
Keyword [en]
substance P, neurotransmitter, tendinopathy, overuse injury, rabbit, tendon cell, Achilles tendon
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-70193ISBN: 978-91-7459-633-5 (print)ISBN: 978-91-7459-634-2 (print)OAI: oai:DiVA.org:umu-70193DiVA: diva2:619908
Public defence
2013-06-05, sal BiA201, Biologihuset, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2013-05-15 Created: 2013-05-07 Last updated: 2013-05-15Bibliographically approved
List of papers
1. Endogenous substance P production in the Achilles tendon increases with loading in an in vivo model of tendinopathy: peptidergic elevation preceding tendinosis-like tissue changes
Open this publication in new window or tab >>Endogenous substance P production in the Achilles tendon increases with loading in an in vivo model of tendinopathy: peptidergic elevation preceding tendinosis-like tissue changes
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2011 (English)In: Journal of Musculoskeletal and Neuronal Interactions - JMNI, ISSN 1108-7161, Vol. 11, no 2, 133-140 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: To quantify the intratendinous levels of substance P (SP) at different stages of overload in an established modelfor Achilles tendinopathy (rabbit). Also, to study the distribution of the SP-receptor, the NK-1R, and the source of SP, in thetendon. 

Methods: Animals were subjected to the overuse protocol for 1, 3 or 6 weeks. One additional group served as unexercisedcontrols. Immunoassay (EIA), immunohistochemistry (IHC), and in situ hybridisation (ISH) were performed.

Results: EIA revealedincreased SP-levels in the Achilles tendon of the exercised limb in all the experimental groups as compared to in thecontrols (statistically significant; p=0.01). A similar trend in the unexercised Achilles tendon was observed but was not statisticallysignificant (p=0.14). IHC and in ISH illustrated reactions of both SP and NK-1R mainly in blood vessel walls, but the receptorwas also found on tenocytes.

Conclusions: Achilles tendon SP-levels are elevated already after 1 week of loading. This showsthat increased SP-production precedes tendinosis, as tendinosis-like changes occur only after a minimum of 3 weeks of exercise,as shown in a recent study using this model. We propose that central neuronal mechanism may be involved as similar trends wereobserved in the contralateral Achilles tendon.

Place, publisher, year, edition, pages
International Society of Musculoskeletal and Neuronal Interactions, 2011
Keyword
Neuropeptides, Neurokinin-1 Receptor, Animal Model, Overuse Injuries, Rabbit
National Category
Sport and Fitness Sciences Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-42299 (URN)000291698500007 ()21625050 (PubMedID)
Available from: 2011-04-07 Created: 2011-04-07 Last updated: 2017-12-11Bibliographically approved
2. Substance P accelerates hypercellularity and angiogenesis in tendon tissue and enhances paratendinitis in response to Achilles tendon overuse in a tendinopathy model
Open this publication in new window or tab >>Substance P accelerates hypercellularity and angiogenesis in tendon tissue and enhances paratendinitis in response to Achilles tendon overuse in a tendinopathy model
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2011 (English)In: British Journal of Sports Medicine, ISSN 0306-3674, E-ISSN 1473-0480, Vol. 45, no 13, 1017-1022 p.Article in journal (Refereed) Published
Abstract [en]

Background Tenocytes produce substance P (SP) and its receptor (neurokinin-1 receptor (NK-1R) is expressed throughout the tendon tissue, expecially in patients with tendinopathy and tissue changes (tendinosis) including hypercellularity and vascular proliferation. Considering the known effects of SP, one might ask whether SP contributes to these canges.

Objectives To test whether development of tendinosislike changes (hypercellularity and angiogenesis) is accelerated during a 1-week course of ecercise with local administration of SP in an establish Achilles tendinopathy model.

Methods Rabbits were subjected to a protocol of Achilles tendon overuse for 1 week, in conjunction with SP injections in the paratenon. Exercised control animals received NaCl injections or no injections, and unexercised, uninjected controls were also used. Tenocyte number and vascular density, as well as paratendinous inflammation, were evaluated. Immunohistochemistry and in sity hybridisation to detect NK-1R were conducted.

Results There was a significant increase in tenocyte number in the SP-injected and NaCl-injected groups compared with both unexercised and exercised, uninjected controls. Tendon blood vessels increased in number in the SP-injected group compared with unexercised controls, a finding not seen in NaCl-injected controls or in uninjected, exercised animals. Paratendinous inflammation was more pronounced in the SP-injected group than in the NaCl controls. NK-1R was detected in blood vessel walls, nerves, inflammatory cells and tenocytes.

Conclusions SP accelerated the development of tendinosis-like changes in the rabbit. Achilles tendon, which supports theories of a potential role of SP in tendinosis development; a fact of clinical interest since SP effects can be effectively blocked. The angiogenic response to SP injections seems related to parateninitis.

Place, publisher, year, edition, pages
Loughborough: British Assoc. of Sport and Medicine, 2011
National Category
Sport and Fitness Sciences
Identifiers
urn:nbn:se:umu:diva-42293 (URN)10.1136/bjsm.2010.082750 (DOI)
Available from: 2011-04-07 Created: 2011-04-07 Last updated: 2017-12-11Bibliographically approved
3. Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation
Open this publication in new window or tab >>Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 11, e27209- p.Article in journal (Refereed) Published
Abstract [en]

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell (R) technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10(-7) M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.

Place, publisher, year, edition, pages
San Francisco, USA: Public Library of Science, 2011
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-50694 (URN)10.1371/journal.pone.0027209 (DOI)000297150900060 ()
Available from: 2011-12-20 Created: 2011-12-19 Last updated: 2017-12-08Bibliographically approved
4. Alpha-2 adrenergic stimulation triggers Achilles tenocyte hypercellularity: comparison between two model systems
Open this publication in new window or tab >>Alpha-2 adrenergic stimulation triggers Achilles tenocyte hypercellularity: comparison between two model systems
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2013 (English)In: Scandinavian Journal of Medicine and Science in Sports, ISSN 0905-7188, E-ISSN 1600-0838, Vol. 23, no 6, 687-696 p.Article in journal (Refereed) Published
Abstract [en]

The histopathology of tendons with painful tendinopathy is often tendinosis, a fibrosis-like condition of unclear pathogenesis characterized by tissue changes including hypercellularity. The primary tendon cells (tenocytes) have been shown to express adrenoreceptors (mainly alpha-2A) as well as markers of catecholamine production, particularly in tendinosis. It is known that adrenergic stimulation can induce proliferation in other cells. The present study investigated the effects of an exogenously administered alpha-2 adrenergic agonist in an established in vivo Achilles tendinosis model (rabbit) and also in an in vitro human tendon cell culture model. The catecholamine producing enzyme tyrosine hydroxylase and the alpha-2A-adrenoreceptor (α(2A) AR) were expressed by tenocytes, and alpha-2 adrenergic stimulation had a proliferative effect on these cells, in both models. The proliferation was inhibited by administration of an α(2A) AR antagonist, and the in vitro model further showed that the proliferative alpha-2A effect was mediated via a mitogenic cell signaling pathway involving phosphorylation of extracellular-signal-regulated kinases 1 and 2. The results indicate that catecholamines produced by tenocytes in tendinosis might contribute to the proliferative nature of the pathology through stimulation of the α(2A) AR, pointing to a novel target for future therapies. The study furthermore shows that animal models are not necessarily required for all aspects of this research.

Place, publisher, year, edition, pages
John Wiley & Sons, 2013
Keyword
tendinopathy, exercise, cytokines, neurotrophins, alpha(2A) adrenoreceptor, Tendinosis, Achilles tendon
National Category
Sport and Fitness Sciences Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-53462 (URN)10.1111/j.1600-0838.2011.01442.x (DOI)22292987 (PubMedID)
Funder
Swedish Research Council, 521-2009-2921Swedish National Centre for Research in Sports, 54/10, P2011-0170
Available from: 2012-03-27 Created: 2012-03-27 Last updated: 2017-12-07Bibliographically approved
5. Akt-mediated anti-apoptotic effects of substance P in Anti-Fas-induced apoptosis of human tenocytes
Open this publication in new window or tab >>Akt-mediated anti-apoptotic effects of substance P in Anti-Fas-induced apoptosis of human tenocytes
2013 (English)In: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 17, no 6, 723-733 p.Article in journal (Refereed) Published
Abstract [en]

Substance P (SP) and its receptor, the neurokinin-1 receptor (NK-1 R), are expressed by human tenocytes, and they are both up-regulated incases of tendinosis, a condition associated with excessive apoptosis. It is known that SP can phosphorylate/activate the protein kinase Akt,which has anti-apoptotic effects. This mechanism has not been studied for tenocytes. The aims of this study were to investigate if Anti-Fastreatment is a good apoptosis model for human tenocytes in vitro, if SP protects from Anti-Fas-induced apoptosis, and by which mechanismsSP mediates an anti-apoptotic response. Anti-Fas treatment resulted in a time- and dose-dependent release of lactate dehydrogenase (LDH), i.e.induction of cell death, and SP dose-dependently reduced the Anti-Fas-induced cell death through a NK-1 R specific pathway. The same trendwas seen for the TUNEL assay, i.e. SP reduced Anti-Fas-induced apoptosis via NK-1 R. In addition, it was shown that SP reduces Anti-Fas-induced decrease in cell viability as shown with crystal violet assay. Protein analysis using Western blot confirmed that Anti-Fas inducescleavage/activation of caspase-3 and cleavage of PARP; both of which were inhibited by SP via NK-1 R. Finally, SP treatment resulted in phosphorylation/activation of Akt as shown with Western blot, and it was confirmed that the anti-apoptotic effect of SP was, at least partly, inducedthrough the Akt-dependent pathway. In conclusion, we show that SP reduces Anti-Fas-induced apoptosis in human tenocytes and that this antiapoptoticeffect of SP is mediated through NK-1 R and Akt-specific pathways.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2013
Keyword
tendinopathy, tendinosis, Neurokinin-1 Receptor, cell death, tachykinin, caspase-3, PARP, tendon cells
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-70190 (URN)10.1111/jcmm.12059 (DOI)000320779100004 ()
Available from: 2013-05-07 Created: 2013-05-07 Last updated: 2017-12-06Bibliographically approved

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