| 1. |
- Backman, Ludvig J, et al.
(författare)
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Alpha-2 adrenergic stimulation triggers Achilles tenocyte hypercellularity : comparison between two model systems
- 2013
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Ingår i: Scandinavian Journal of Medicine and Science in Sports. - : John Wiley & Sons. - 0905-7188 .- 1600-0838. ; 23:6, s. 687-696
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Backman, Ludvig, et al.
(författare)
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Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation
- 2011
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Ingår i: PLOS ONE. - San Francisco, USA : Public Library of Science. - 1932-6203. ; 6:11, s. e27209-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 4. |
- Fong, Gloria, et al.
(författare)
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Human tenocytes are stimulated to proliferate by acetylcholine through an EGFR signalling pathway
- 2013
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Ingår i: Cell and Tissue Research. - : Springer-Verlag New York. - 0302-766X .- 1432-0878. ; 351:3, s. 465-475
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Fong, Gloria, 1986-
(författare)
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Influence of neuromodulators and mechanical loading on pathological cell and tissue characteristics in tendinosis
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
- Fong, Gloria, et al.
(författare)
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Substance P enhances collagen remodeling and MMP-3 expression by human tenocytes
- 2013
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Ingår i: Journal of Orthopaedic Research. - HOBOKEN, NJ, USA : Wiley-Blackwell. - 0736-0266 .- 1554-527X. ; 31:1, s. 91-98
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Fong, Gloria, et al.
(författare)
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The Effects of Substance P and Acetylcholine on Human Tenocyte Proliferation Converge Mechanistically via TGF-β1
- 2017
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:3
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Tidskriftsartikel (refereegranskat)abstract
- Previous in vitro studies on human tendon cells (tenocytes) have demonstrated that the exogenous administration of substance P (SP) and acetylcholine (ACh) independently result in tenocyte proliferation, which is a prominent feature of tendinosis. Interestingly, the possible link between SP and ACh has not yet been explored in human tenocytes. Recent studies in other cell types demonstrate that both SP and ACh independently upregulate TGF-β1 expression via their respective receptors, the neurokinin 1 receptor (NK-1R) and muscarinic ACh receptors (mAChRs). Furthermore, TGF-β1 has been shown to downregulate NK-1R expression in human keratocytes. The aim of this study was to examine if TGF-β1 is the intermediary player involved in mediating the proliferative pathway shared by SP and ACh in human tenocytes. The results showed that exogenous administration of SP and ACh both caused significant upregulation of TGF-β1 at the mRNA and protein levels. Exposing cells to TGF-β1 resulted in increased cell viability of tenocytes, which was blocked in the presence of the TGFβRI/II kinase inhibitor. In addition, the proliferative effects of SP and ACh on tenocytes were reduced by the TGFβRI/II kinase inhibitor; this supports the hypothesis that the proliferative effects of these signal substances are mediated via the TGF-β axis. Furthermore, exogenous TGF-β1 downregulated NK-1R and mAChRs expression at both the mRNA and protein levels, and these effects were negated by simultaneous exposure to the TGFβRI/II kinase inhibitor, suggesting a negative feedback loop. In conclusion, the results indicate that TGF-β1 is the intermediary player through which the proliferative actions of both SP and ACh converge mechanistically.
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| 8. |
- Tiwari, Agnes, et al.
(författare)
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Evaluating the Chinese Revised Controlling Behaviors Scale
- 2015
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Ingår i: Journal of Interpersonal Violence. - : SAGE Publications. - 0886-2605 .- 1552-6518. ; 30:2, s. 314-332
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Tidskriftsartikel (refereegranskat)abstract
- The present study evaluated the utility of the Chinese version of the Revised Controlling Behaviors Scale (C-CBS-R) as a measure of controlling behaviors in violent Chinese intimate relationships. Using a mixed-methods approach, in-depth, individual interviews were conducted with 200 Chinese women survivors to elicit qualitative data about their personal experiences of control in intimate relationships. The use of controlling behaviors was also assessed using the C-CBS-R. Interview accounts suggested that the experiences of 91 of the women were consistent with the description of coercive control according to Dutton and Goodman's conceptualization of coercion. Using the split-half validation procedure, a receiver operating characteristics (ROC) curve analysis was conducted with the first half of the sample. The area under the curve (AUC) for using the C-CBS-R to identify high control was .99, and the cutoff score of 1.145 maximized both sensitivity and specificity. Applying the cutoff score to the second half gave a sensitivity of 96% and a specificity of 95%. Overall, the C-CBS-R has demonstrated utility as a measure of controlling behaviors with a cutoff score for distinguishing high from low levels of control in violent Chinese intimate relationships.
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