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

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© 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.

Human Tenocytes Are Stimulated to Proliferate by Acetylcholine Through an Egfr Signalling Pathway

“…1a) and tenomodulin (Fig. 1b) in the passages and serum concentrations used for experiments, thereby indicating a fibroblastic phenotype (tenocytes), as previously described for this model (Backman et al 2011). The presence of scleraxis protein was also confirmed by using Western blot (Fig.…”

“…Biopsies were isolated and cultured as previously described (Backman et al 2011). Briefly, tendon tissue biopsies from the lateral mid-portion of the Achilles tendon of healthy donors were enzymatically digested and the cells were cultured in D-MEM supplemented with 10% fetal bovine serum (FBS; Invitrogen, Grand Island, N.Y., USA; code no.…”

“…Crystal violet dye was used to measure cell viability as previously described (Backman et al 2011). Briefly, 1.5×10 5 cells/well were seeded in a 6-well plate and allowed to adhere overnight before serum starvation.…”

“…As previously described (Backman et al 2011), total RNA was isolated by using an RNeasy kit (Qiagen, Hilden, Germany; code no. 74106) and then 1 μg of this RNA was reversed-transcribed into cDNA by using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems [ABI], Warrington, Cheshire, UK; code no.…”

Neuronal pathways in tendon healing and tendinopathy - update

“…SP has also been reported to stimulate proliferation of fibroblasts (143) and endothelial cells, as well as the production of transforming growth factor beta in fibroblasts. Hence, SP may well contribute to the morphologic changes observed in early tendinopathic patients, that is, tenocyte transformation, hypercellularity, and presumably neovascularization, in conjunction with mechanical loading (37,144), and may therefore precede tendinosis.…”