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Influence of neuromodulators and mechanical loading on pathological cell and tissue characteristics in tendinosis
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Patrik Danielson)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Betydelsen av neuromodulatorer och mekanisk belastning för cell- och vävnadsförändringar vid tendinos (Swedish)
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.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2017. , 60 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1882
Keyword [en]
substance P, acetylcholine, transforming growth factor, neuromodulators, mechanical loading, tendinosis
National Category
Cell and Molecular Biology
Research subject
Human Anatomy
Identifiers
URN: urn:nbn:se:umu:diva-131390ISBN: 978-91-7601-666-4 (electronic)OAI: oai:DiVA.org:umu-131390DiVA: diva2:1073929
Public defence
2017-03-10, KBC-huset, sal KB.E3.01, Umeå universitet, Umeå, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 521-2013-2612Swedish Society of Medicine, SLS-504541Swedish National Centre for Research in Sports, P2013-0055
Available from: 2017-02-17 Created: 2017-02-13 Last updated: 2017-03-16Bibliographically approved
List of papers
1. Human tenocytes are stimulated to proliferate by acetylcholine through an EGFR signalling pathway
Open this publication in new window or tab >>Human tenocytes are stimulated to proliferate by acetylcholine through an EGFR signalling pathway
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2013 (English)In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 351, no 3, 465-475 p.Article in journal (Refereed) Published
Abstract [en]

Studies of human patellar and Achilles tendons have shown that primary tendon fibroblasts (tenocytes) not only have the capacity to produce acetylcholine (ACh) but also express muscarinic ACh receptors (mAChRs) through which ACh can exert its effects. In patients with tendinopathy (chronic tendon pain) with tendinosis, the tendon tissue is characterised by hypercellularity and angiogenesis, both of which might be influenced by ACh. In this study, we have tested the hypothesis that ACh increases the proliferation rate of tenocytes through mAChR stimulation and have examined whether this mechanism operates via the extracellular activation of the epidermal growth factor receptor (EGFR), as shown in other fibroblastic cells. By use of primary human tendon cell cultures, we identified cells expressing vimentin, tenomodulin and scleraxis and found that these cells also contained enzymes related to ACh synthesis and release (choline acetyltransferase and vesicular acetylcholine transporter). The cells furthermore expressed mAChRs of several subtypes. Exogenously administered ACh stimulated proliferation and increased the viability of tenocytes in vitro. When the cells were exposed to atropine (an mAChR antagonist) or the EGFR inhibitor AG1478, the proliferative effect of ACh decreased. Western blot revealed increased phosphorylation, after ACh stimulation, for both EGFR and the extracellular-signal-regulated kinases 1 and 2. Given that tenocytes have been shown to produce ACh and express mAChRs, this study provides evidence of a possible autocrine loop that might contribute to the hypercellularity seen in tendinosis tendon tissue.

Place, publisher, year, edition, pages
Springer-Verlag New York, 2013
Keyword
Muscarinic acetylcholine receptors, Tendinopathy, Tendinosis, Choline acetyltransferase, Vesicular acetylcholine transporter, Atropine, Non-neuronal acetylcholine, Human
National Category
Surgery
Identifiers
urn:nbn:se:umu:diva-67794 (URN)10.1007/s00441-012-1530-5 (DOI)000315490400011 ()
Available from: 2013-04-05 Created: 2013-04-03 Last updated: 2017-02-16Bibliographically approved
2. 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, 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: 2017-02-13Bibliographically approved
3. Substance P enhances collagen remodeling and MMP-3 expression by human tenocytes
Open this publication in new window or tab >>Substance P enhances collagen remodeling and MMP-3 expression by human tenocytes
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2013 (English)In: Journal of Orthopaedic Research, ISSN 0736-0266, E-ISSN 1554-527X, Vol. 31, no 1, 91-98 p.Article in journal (Refereed) Published
Abstract [en]

The loss of collagen organization is considered a hallmark histopathologic feature of tendinosis. At the cellular level, tenocytes have been shown to produce signal substances that were once thought to be restricted to neurons. One of the main neuropeptides implicated in tendinosis, substance P (SP), is known to influence collagen organization, particularly after injury. The aim of this study was to examine the influence of SP on collagen remodeling by primary human tendon cells cultured in vitro in three-dimensional collagen lattices. We found that SP stimulation led to an increased rate of collagen remodeling mediated via the neurokinin-1 receptor (NK-1 R), the preferred cell receptor for SP. Gene expression analysis showed that SP stimulation resulted in significant increases in MMP3, COL3A1 and ACTA2 mRNA levels in the collagen lattices. Furthermore, cyclic tensile loading of tendon cell cultures along with the administration of exogenous SP had an additive effect on MMP3 expression. Immunoblotting confirmed that SP increased MMP3 protein levels via the NK-1 R. This study indicates that SP, mediated via NK-1 R, increases collagen remodeling and leads to increased MMP3 mRNA and protein expression that is further enhanced by cyclic mechanical loading.

Place, publisher, year, edition, pages
HOBOKEN, NJ, USA: Wiley-Blackwell, 2013
Keyword
tendinopathy, tendinosis, collagen, myofibroblasts, neurokinin-1 receptor
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-63758 (URN)10.1002/jor.22191 (DOI)000311568700013 ()
Available from: 2013-01-10 Created: 2013-01-07 Last updated: 2017-02-16Bibliographically approved
4. The Effects of Substance P and Acetylcholine on Human Tenocyte Proliferation Converge Mechanistically via TGF-β1
Open this publication in new window or tab >>The Effects of Substance P and Acetylcholine on Human Tenocyte Proliferation Converge Mechanistically via TGF-β1
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-131396 (URN)
Available from: 2017-02-13 Created: 2017-02-13 Last updated: 2017-02-13

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