Presence of substance P and the neurokinin-1 receptor in tenocytes of the human Achilles tendon
Introduction
Chronically painful Achilles tendon tendinosis (tendinopathy) is verified by radiological and/or histological findings of abnormal tissue structure, hypercellularity, and neovascularization [1]. A similar condition can affect the patellar tendon. The etiology of Achilles and patellar tendinosis is largely unknown.
In recent studies on Achilles and patellar tendinosis, the cells within the tendon tissue (the tenocytes) were unexpectedly found to express enzymes related to the production of the neurotransmitters acetylcholine and catecholamines [2], [3], [4], [5], [6], [7]. The tenocytes were also found to express the vesicular glutamate transporter VGluT2 suggesting that glutamate might be locally produced and released by the tenocytes [8]. Furthermore, the tenocytes were found to be equipped with muscarinic and adrenergic receptors [2], [3], [4], [5], [7]. NMDAR1 glutamate receptors have previously been observed in tendon tissue [9]. Apart from expressing the required enzymes and receptors for the cholinergic and adrenergic pathways, the tenocytes of tendinosis samples have been shown to over-express these factors in comparison to normal tendons [2], [3], [4], [5], [7]. The levels of VGluT2 expression in these cells [8], and the levels of free glutamate [9], were also higher in tendinosis tendons than in normal tendons. This enhanced local production/function of the transmitters may be of significance in the development of, or response to, tendinosis [2], [3], [4], [5], [7], [8], [9].
A neuropeptide that is known to be involved in a number of important functions is substance P (SP). Classically SP is confined to sensory neurons. However, it is now also described that SP is expressed by non-neuronal cells (e.g. [10], [11]). This neuropeptide has been found not only to be related to pain transmission but also to have effects on cell growth and angiogenesis [12], to promote vasodilatation and increased vascular permeability [13], [14], and to exhibit healing effects [15], [16], [17].
The occurrence of a SP innervation has been observed for both normal and tendinosis Achilles tendons of man [18]. Most of the SP innervation was observed in the paratendinous loose connective tissue, i.e. the tissue surrounding the tendon tissue proper. That includes the paratendinous loose connective tissue at the ventral side of the Achilles tendon [19], a region where the pain symptoms predominate. It is not known if there is also a local SP production in the tenocytes within the tendon tissue proper. This is of particular relevance to clarify, as SP is reported to have healing and growth-promoting functions concerning tendons [20], and influences on the organization of tendon tissue in tendinosis [21]. Of further relevance is the fact that the level of SP innervation within the tendon tissue proper is sparse [18].
Substance P binds with highest affinity to the neurokinin-1 receptor (NK-1 R) [22]. The NK-1 R belongs to the family of G-protein-coupled receptors and has 407 amino acids [22], [23]. The presence of the NK-1 R in tenocytes of both Achilles and patellar tendons has been briefly described in a study focusing on the blood vessels and the nerve fascicles [24]. In a following study, NK-1 R immunoexpression was shown for nerve fascicles, and particularly for the walls of blood vessels, in the paratendinous loose connective tissue on the ventral side of the Achilles tendon [19]. The details in NK-1 R immunoexpression levels for tenocytes are unknown.
So far, there is no information at all concerning SP and NK-1 R expressions at the mRNA level for tendon tissue. For this reason, and based on what is described above, it is of interest to clarify if the tenocytes in tendinosis tendons can produce SP, and if there are changes in the patterns of NK-1 R immunoexpression in tenocytes in response to tendinosis. Therefore, in the present study, we examined the patterns of expression of SP and the NK-1 R for tenocytes in tendinosis and normal Achilles tendons. Both immunofluorescence staining and in situ hybridization were applied.
Section snippets
Individuals
This investigation included tissue samples from a total of 27 individuals (mean age: 47 years; range: 26–67 years) either suffering from chronic painful mid-portion Achilles tendinosis (“tendinosis tendons”) or being without symptoms (“normal tendons”). Samples from all of these individuals were examined immunohistochemically. They were processed in a chemically fixed state and/or in a chemically unfixed state.
The tendinosis group consisted of nine males and eleven females (males mean age:
General observations
Nerve fascicles were frequently observed in the paratendinous connective tissue. Such fascicles sometimes also occurred in the connective tissue spaces within the tendon tissue proper. Blood vessels regularly occurred in these connective tissues, and were a particularly prominent feature in certain tendinosis tendons. The tendon cells (tenocytes) were generally more frequent in tendinosis tendons than in normal tendons. These morphologic features correspond to those found in previous studies on
Discussion
The results of the present study show that tenocytes of Achilles tendons display expression of SP and NK-1 R. Expression at both protein and mRNA levels was shown for the NK-1 R, whilst SP was demonstrated at the mRNA level. The labeling was detected for a subpopulation of the tenocytes, the semi-quantitative estimations suggesting higher expression levels of both NK-1 R and SP in tendinosis tendons compared with normal tendons.
Acknowledgements
The authors thank Ms. Ulla Hedlund for excellent technical services. Financial support was obtained by the Faculty of Medicine at Umeå University, the Swedish National Centre for Research in Sports, Muscle Research Fund North, the County Council of Västerbotten, and the J.C. Kempe and Seth M. Kempe Memorial Foundations, Örnsköldsvik.
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