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.
Highlights
In recent years, increasing attention has been devoted to the non-neuronal expression of signal substances traditionally associated with neurons and the potential roles of such substances in various pathological conditions
The vast majority of cells in the primary cultures were immunopositive for vimentin, scleraxis (Fig. 1a) and tenomodulin (Fig. 1b) in the passages and serum concentrations used for experiments, thereby indicating a fibroblastic phenotype, as previously described for this model (Backman et al 2011)
The present study shows that human Achilles tenocytes express enzymes related to ACh synthesis/release
Summary
In recent years, increasing attention has been devoted to the non-neuronal expression of signal substances traditionally associated with neurons and the potential roles of such substances in various pathological conditions. A physiological role of nonneuronal ACh might explain the widespread presence of ACh receptors, such as muscarinic receptors (mAChRs), in tissues not innervated by cholinergic neurons (Wessler et al 1998). We have demonstrated that mAChRs of subtype M2 (M2Rs) are expressed in blood vessels, nerves and primary fibroblastic cells (tenocytes) in both Achilles (Bjur et al 2008) and patellar (Danielson et al 2006) tendons of man, whereas cholinergic innervation is scarce or absent. A striking finding in this regard is the suggested increased production and release of ACh in tendinosis tissue, as evidenced by a higher expression of ChAT and the vesicular ACh transporter (VAChT) in tenocytes (Danielson et al 2006, 2007; Bjur et al 2008)
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