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Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation
Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB), Anatomi. Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin.ORCID-id: 0000-0002-6091-3982
Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB), Anatomi.
Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB), Anatomi.
Vancouver Coastal Health and Research Institute, University of British Columbia, Vancouver.
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2011 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, nr 11, s. e27209-Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
San Francisco, USA: Public Library of Science , 2011. Vol. 6, nr 11, s. e27209-
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
URN: urn:nbn:se:umu:diva-50694DOI: 10.1371/journal.pone.0027209ISI: 000297150900060OAI: oai:DiVA.org:umu-50694DiVA, id: diva2:468139
Tillgänglig från: 2011-12-20 Skapad: 2011-12-19 Senast uppdaterad: 2019-02-22Bibliografiskt granskad
Ingår i avhandling
1. Neuropeptide and catecholamine effects on tenocytes in tendinosis development: studies on two model systems with focus on proliferation and apoptosis
Öppna denna publikation i ny flik eller fönster >>Neuropeptide and catecholamine effects on tenocytes in tendinosis development: studies on two model systems with focus on proliferation and apoptosis
2013 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå Universitet, 2013. s. 80
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1572
Nyckelord
substance P, neurotransmitter, tendinopathy, overuse injury, rabbit, tendon cell, Achilles tendon
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-70193 (URN)978-91-7459-633-5 (ISBN)978-91-7459-634-2 (ISBN)
Disputation
2013-06-05, sal BiA201, Biologihuset, Umeå universitet, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2013-05-15 Skapad: 2013-05-07 Senast uppdaterad: 2019-02-22Bibliografiskt granskad
2. Influence of neuromodulators and mechanical loading on pathological cell and tissue characteristics in tendinosis
Öppna denna publikation i ny flik eller fönster >>Influence of neuromodulators and mechanical loading on pathological cell and tissue characteristics in tendinosis
2017 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Alternativ titel[sv]
Betydelsen av neuromodulatorer och mekanisk belastning för cell- och vävnadsförändringar vid tendinos
Abstract [en]

Background: Tendinosis is a painful chronic, degenerative condition characterized by objective changes in the tissue structure of a tendon. Hallmark features in tendinosis tendons include increased number of cells (hypercellularity), extracellular matrix (ECM) degradation and disorganized collagen. The progression of these pathological changes seen in tendinosis is neither well characterized nor fully understood.

Studies have suggested that there are biochemical and mechanical elements involved in tendinosis. From a biochemical perspective, studies have shown that the tendon cells, tenocytes, produce a number of neuronal signal substances/neuromodulators, such as substance P (SP) and acetylcholine (ACh), traditionally thought to be confined to the nervous system. Furthermore, it has been shown that the expression of these neuromodulators is elevated in tendinosis tendons as compared to normal healthy tendons. Interestingly, studies on other tissue types have revealed that both SP and ACh can induce tissue changes seen in tendinosis, such as hypercellularity and collagen disorganization. From a mechanical angle, it has been suggested that overload of tendons, including extensive strain on the primary tendon cells (tenocytes), causes the degenerative processes associated with tendinosis. In vivo studies have shown that in overloaded tendons, the presence of neuromodulators is elevated, not least SP, which also precedes the development of the tissue changes seen in tendinosis. This further supports the importance of combining biochemical factors and mechanical factors in the pathogenesis of tendinosis.

Hypotheses: In this thesis project, we hypothesize: 1) that neuromodulators, such as SP and ACh when stimulating their preferred receptors, the neurokinin 1 (NK-1 R) and muscarinic receptors (mAChRs), respectively, can cause increased tenocyte proliferation; 2) that the effects of SP and ACh on tenocyte proliferation converge mechanistically via a shared signalling pathway; 3) that mechanical loading of tenocytes results in increased production of SP by the tenocytes; and 4) that SP enhances collagen remodelling by tenocytes via NK-1 R.

Model system: In vitro studies offer insight into the function of healthy tendon matrix and the etiology of tendinopathy. Using a cell culture model of human primary tendon cells, highly controlled experiments were performed in this thesis project to study a subset of biological and mechanical parameters that are implicated in tendinosis. The FlexCell® Tension System was used to study the influence of mechanical loading on tenocytes. As well, a collagen gel contraction assay was used to examine the intrinsic ability of tenocytes to reorganise type I collagen matrices under the influence of the neuromodulator SP.

Results: The studies showed that exogenous administration of SP and ACh results in increased tenocyte proliferation that is mediated via activation of the ERK1/2 mitogenic pathway when the preferred receptors of SP and ACh, the NK-1 R and mAChRs, respectively, are stimulated. Furthermore, the studies resulted in the novel finding that SP and ACh both converge mechanistically via transforming growth factor (TGF)-β1 and that a negative feedback mechanism is present in which TGF-β1 downregulates the expression of mAChRs and NK-1 R. The studies also showed that SP can increase collagen remodelling and upregulate expression of genes related to tendinosis. Finally, it was established that tenocytes are mechanoresponsive by showing that cyclic mechanical loading increases the expression of SP by human tenocytes.

Conclusions: This thesis work concludes that stimulation of NK-1 R and mAChRs results in proliferation of human tenocytes, which both involve the ERK1/2 signalling pathway. It also shows that SP and ACh converge mechanistically via TGF-β1 in their contribution to tenocyte proliferation. The role of hypercellularity in tendinosis tissue is unknown. Possibly, it has different roles at different stages of the disease. The findings also show that SP increases collagen remodelling, suggesting that increased SP not only results in hypercellularity but also contributes to the collagen morphology in tendinosis.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2017. s. 60
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1882
Nyckelord
substance P, acetylcholine, transforming growth factor, neuromodulators, mechanical loading, tendinosis
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
anatomi
Identifikatorer
urn:nbn:se:umu:diva-131390 (URN)978-91-7601-666-4 (ISBN)
Disputation
2017-03-10, KBC-huset, sal KB.E3.01, Umeå universitet, Umeå, 13:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
Vetenskapsrådet, 521-2013-2612Svenska läkaresällskapet, SLS-504541Centrum för Idrottsforskning, P2013-0055
Tillgänglig från: 2017-02-17 Skapad: 2017-02-13 Senast uppdaterad: 2019-02-22Bibliografiskt granskad

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