Ludvig J. Backman

Substance P Is a Mechanoresponsive, Autocrine Regulator of Human Tenocyte Proliferation

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© 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.

Low range of ankle dorsiflexion predisposes for patellar tendinopathy in junior elite basketball players : a 1-year prospective study

Background: Patellar tendinopathy (PT) is one of the most common reasons for sport-induced pain of the knee. Low ankle dorsiflexion range might predispose for PT because of load-bearing compensation in the patellar tendon. Purpose: The purpose of this 1-year prospective study was to analyze if a low ankle dorsiflexion range increases the risk of developing PT for basketball players. Study Design: Cohort study (prognosis); Level of evidence, 2. Methods: Ninety junior elite basketball players were examined for different characteristics and potential risk factors for PT, including ankle dorsiflexion range in the dominant and nondominant leg. Data were collected over a 1-year period and follow-up, including reexamination, was made at the end of the year. Results: Seventy-five players met the inclusion criteria. At the follow-up, 12 players (16.0%) had developed unilateral PT. These players were found to have had a significantly lower mean ankle dorsiflexion range at baseline than the healthy players, with a mean difference of −4.7° (P = .038) for the dominant limb and −5.1° (P = .024) for the nondominant limb. Complementary statistical analysis showed that players with dorsiflexion range less than 36.5° had a risk of 18.5% to 29.4% of developing PT within a year, as compared with 1.8% to 2.1% for players with dorsiflexion range greater than 36.5°. Limbs with a history of 2 or more ankle sprains had a slightly less mean ankle dorsiflexion range compared to those with 0 or 1 sprain (mean difference, −1.5° to −2.5°), although this was only statistically significant for nondominant legs. Conclusion: This study clearly shows that low ankle dorsiflexion range is a risk factor for developing PT in basketball players. In the studied material, an ankle dorsiflexion range of 36.5° was found to be the most appropriate cutoff point for prognostic screening. This might be useful information in identifying at-risk individuals in basketball teams and enabling preventive actions. A history of ankle sprains might contribute to reduced ankle dorsiflexion range.

Substance P accelerates hypercellularity and angiogenesis in tendon tissue and enhances paratendinitis in response to Achilles tendon overuse in a tendinopathy model

Background Tenocytes produce substance P (SP), and its receptor (neurokinin-1 receptor (NK-1R)) is expressed throughout the tendon tissue, especially 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 changes. Objectives To test whether development of tendinosis-like changes (hypercellularity and angiogenesis) is accelerated during a 1-week course of exercise with local administration of SP in an established 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 situ 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 paratendinitis.

Tendinopathy: Update on Pathophysiology

Synopsis Tendinopathy has become the accepted term to describe a spectrum of changes that occur in damaged and/or diseased tendons. Over the past 2 decades, there have been new insights into tendon pathophysiology of relevance to clinicians, including (1) better characterization of the overuse injury process and the resultant structural and functional disruption in chronically painful tendons, (2) improved understanding of the pathomechanics associated with chronic tendon injury, and (3) greater knowledge about the influence of lifestyle factors and drugs on tendon pathology. The implications of these new insights are discussed. J Orthop Sports Phys Ther 2015;45(11):833-841. Epub 21 Sep 2015. doi:10.2519/jospt.2015.5884.

Substance P enhances collagen remodeling and MMP-3 expression by human tenocytes.

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.

Akt‐mediated anti‐apoptotic effects of substance P in Anti‐Fas‐induced apoptosis of human tenocytes

Substance P (SP) and its receptor, the neurokinin‐1 receptor (NK‐1 R), are expressed by human tenocytes, and they are both up‐regulated in cases 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‐Fas treatment is a good apoptosis model for human tenocytes in vitro, if SP protects from Anti‐Fas‐induced apoptosis, and by which mechanisms SP 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 trend was 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 induces cleavage/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, induced through the Akt‐dependent pathway. In conclusion, we show that SP reduces Anti‐Fas‐induced apoptosis in human tenocytes and that this anti‐apoptotic effect of SP is mediated through NK‐1 R and Akt‐specific pathways.

Mutations in Collagen, Type XVII, Alpha 1 (COL17A1) Cause Epithelial Recurrent Erosion Dystrophy (ERED)

Corneal dystrophies are a clinically and genetically heterogeneous group of inherited disorders that bilaterally affect corneal transparency. They are defined according to the corneal layer affected and by their genetic cause. In this study, we identified a dominantly inherited epithelial recurrent erosion dystrophy (ERED)‐like disease that is common in northern Sweden. Whole‐exome sequencing resulted in the identification of a novel mutation, c.2816C>T, p.T939I, in the COL17A1 gene, which encodes collagen type XVII alpha 1. The variant segregated with disease in a genealogically expanded pedigree dating back 200 years. We also investigated a unique COL17A1 synonymous variant, c.3156C>T, identified in a previously reported unrelated dominant ERED‐like family linked to a locus on chromosome 10q23‐q24 encompassing COL17A1. We show that this variant introduces a cryptic donor site resulting in aberrant pre‐mRNA splicing and is highly likely to be pathogenic. Bi‐allelic COL17A1 mutations have previously been associated with a recessive skin disorder, junctional epidermolysis bullosa, with recurrent corneal erosions being reported in some cases. Our findings implicate presumed gain‐of‐function COL17A1 mutations causing dominantly inherited ERED and improve understanding of the underlying pathology.

HUMAN TENOCYTES ARE STIMULATED TO PROLIFERATE BY ACETYLCHOLINE THROUGH AN EGFR SIGNALLING PATHWAY

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.

Ciliary Neurotrophic Factor Promotes the Migration of Corneal Epithelial Stem/progenitor Cells by Up-regulation of MMPs through the Phosphorylation of Akt

The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt), and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway.

Substance P reduces TNF-α-induced apoptosis in human tenocytes through NK-1 receptor stimulation

Background It has been hypothesised that an upregulation of the neuropeptide substance P (SP) and its preferred receptor, the neurokinin-1 receptor (NK-1 R), is a causative factor in inducing tenocyte hypercellularity, a characteristic of tendinosis, through both proliferative and antiapoptotic stimuli. We have demonstrated earlier that SP stimulates proliferation of human tenocytes in culture. Aim The aim of this study was to investigate whether SP can mediate an antiapoptotic effect in tumour necrosis factor-α (TNF-α)-induced apoptosis of human tenocytes in vitro. Results A majority (approximately 75%) of tenocytes in culture were immunopositive for TNF Receptor-1 and TNF Receptor-2. Exposure of the cells to TNF-α significantly decreased cell viability, as shown with crystal violet staining. TNF-α furthermore significantly increased the amount of caspase-10 and caspase-3 mRNA, as well as both BID and cleaved-poly ADP ribosome polymerase (c-PARP) protein. Incubation of SP together with TNF-α resulted in a decreased amount of BID and c-PARP, and in a reduced lactate dehydrogenase release, as compared to incubation with TNF-α alone. The SP effect was blocked with a NK-1 R inhibitor. Discussion This study shows that SP, through stimulation of the NK-1 R, has the ability to reduce TNF-α-induced apoptosis of human tenocytes. Considering that SP has previously been shown to stimulate tenocyte proliferation, the study confirms SP as a potent regulator of cell-turnover in tendon tissue, capable of stimulating hypercellularity through different mechanisms. This gives further support for the theory that the upregulated amount of SP seen in tendinosis could contribute to hypercellularity.