Indeed, significant inflammatory infiltrates in the skin are often limited to very early disease stages, and even in these early disease stages meaningful infiltrates can only be found in perivascular areas. There is also increasing evidence that the molecular expression pattern in SSc skin is extremely heterogeneous, and that only smaller subsets of patients express increased levels of inflammatory molecules [10]. Thus, unselective immunosuppressives might be useful for smaller subsets of SSc patients, while the large majority of patients might need different therapeutic approaches. In this regard, mechanisms leading to the increased synthesis of extracellular matrix proteins in fibroblasts are of particular interest for targeted therapies.
Remarkable breakthrough findings have been obtained regarding the identification of key molecules, key cellular mechanisms, and key intracellular signaling cascades, which mediate the perpetuation of fibrosis. These findings have true translational implications, because the modifiers of these key mediators and key mechanisms are often in clinical use in other diseases such as cancer. Among these key molecules, recent reports showed an important role of the 5-hydroxytryptamine (5-HT: serotonin) pathway in fibrosis [11]. Here, we focus on the serotonin pathway as a novel target in the treatment of SSc and summarize these recent results as an example for a preclinical characterization of molecular targets in SSc.
Microvascular damage is one of the earliest features in the pathogenesis of SSc leading to a progressive loss of capillaries [12]. Microvascular injury precedes clinically detectable tissue fibrosis in SSc. However, molecular links between microvascular damage and the induction of tissue fibrosis have not been established. One possible link could be 5-HT, because the microvascular damage with exposure of subendothelial connective tissue activates platelets and activated platelets release large amounts of 5-HT. Based on these considerations and the fact that 5-HT is elevated in the blood of SSc patients [13–15], we hypothesized that 5-HT signaling might be involved in the process of fibrosis in SSc [11].
First, we investigated, whether 5-HT is able to induce the release of extracellular matrix proteins in SSc. Primary dermal fibroblasts from SSc patients and healthy subjects, which were stimulated with 5-HT, increased the mRNA of different extracellular matrix proteins such as collagen type I alpha 1 (COL1A1), collagen type I alpha 2 (COL1A2) and fibronectin-1 in dose-dependent manner. Similarly, the release of collagen protein was also increased by 5-HT stimulation. Doses used for the stimulations were in the range of those detected in biological fluids.
There are seven 5-HT receptors, 5-HT1 to 5-HT7, and the cellular effects of 5-HT are mediated by these receptors. The identification of those receptors, which play a crucial role in fibrosis, was necessary, because receptor antagonists often show more efficacy and safety than ligand antagonist. Thus, we next investigated, which receptor specifically mediates the profibrotic effects of 5-HT. We observed that three different 5-HT receptors were expressed by dermal fibroblasts: 5-HT1B, 5-HT2A, and 5-HT2B. The mRNA levels of 5-HT2B were up regulated in SSc fibroblasts as compared with healthy controls (143 ± 17%), while 5-HT1B and 5-HT2A were not different. Inhibition of 5-HT1 by selective chemical inhibitors showed no reduction of the stimulatory effects of 5-HT on extracellular matrix production in SSc fibroblasts. In contrast, inhibition of 5-HT2 decreased mRNA levels of COL 1A1, COL 1A2 and fibronectin-1 and also decreased the release of collagen protein. Additional inhibition experiments using chemical inhibitors as well as siRNA approaches revealed that this effect was specific for 5-HT2B, but not for 5-HT2A. These results suggested that 5-HT2B plays crucial role for the synthesis of extracellular matrix proteins in dermal fibroblasts.
Immunohistochemistry in fibrotic skin biopsies of SSc patients and normal skin of healthy individuals backed up these results. Expression of 5-HT2B was strongly increased in fibrotic tissue as compared with unaffected tissue from healthy controls. Double staining with the fibroblast-specific marker prolyl-4-hydroxylase-beta confirmed that 5-HT2B was mostly expressed by dermal fibroblasts, and the large majority of fibroblasts stained positive for 5-HT2B in fibrotic tissue, but not in controls.
The profibrotic effects of 5-HT/5-HT2B could be mediated via direct increase of collagen mRNA transcription or indirectly via induction of a second mediator. We suspected indirect mechanisms, because the effects of 5-HT in the COL1A2 reporter assay were delayed compared to the effects of transforming growth factor-β (TGF-β), which is a well known key player in the pathogenesis of fibrosis [16]. Thus, we investigated, whether TGF-β itself might be the second mediator of 5-HT signalling. Interestingly, 5-HT increased dose-dependently mRNA levels of TGF-β1 in SSc fibroblasts. Furthermore, 5-HT induced in a time-dependent manner the nuclear levels of phospho-Smad3, the typical intracellular mediator of TGF-β signalling. To evaluate whether TGF-β is necessary for the profibrotic effect of 5-HT, we cultured SSc fibroblasts with neutralizing antibodies against TGF-β1. Inhibition of TGF-β1 completely abrogated the profibrotic effects of 5-HT on mRNA expression of COL1A1, COL 1A2 and fibronectin-1.
Next, we aimed to demonstrate that these in vitro results reflect in vivo situations by using the mouse model of bleomycin-induced dermal fibrosis. Injection of bleomycin potently stimulated the expression of 5-HT2B and induced dermal fibrosis. The 5-HT2 inhibitors terguride and cyproheptadine as well as the selective 5-HT2B inhibitor SB 204741 [17–20] efficiently prevented bleomycine-induced dermal thickening. Collagen content and myofibroblast counts were also reduced dose-dependently by inhibition of 5-HT2B. The antifibrotic effects of 5-HT2 inhibition were further tested in a therapeutic approach using a modification of the bleomycin model. Dermal thickening decreased by 78 ± 4% in mice treated with terguride for the last 3 weeks compared with placebo treated mice. Finally, we used 5-HT2B -deficient mice to confirm the role of 5-HT2B in experimental fibrosis. 5-HT2B-/- mice were almost completely protected from bleomycin-induced dermal fibrosis, although no spontaneous histological changes or differences in dermal thickness were observed in untreated 5-HT2B-/- mice.
There is no single animal model that completely covers the different aspects in the pathogenesis of SSc [21]. For example, in the bleomycin model, fibrosis is triggered by intense inflammatory infiltrates in the skin. As outlined above, inflammatory infiltrates are present in early inflammatory stages of human SSc, but are not a feature of later disease stages. Thus, for the identification of potential molecular targets for therapy, testing in at least one additional model is recommended [22]. We therefore assessed the role of 5-HT2B in a less inflammation-dependent mouse model of fibrosis, the tight skin 1 (Tsk-1) model. As in human SSc and experimental bleomycin-induced fibrosis, 5-HT2B was overexpressed in skin sections of Tsk-1 mice. Hypodermal thickening, collagen content, and differentiation of resting fibroblasts into myofibroblasts were significantly reduced in Tsk-1 mice upon treatment with the 5-HT2B inhibitor SB 204741. Similarly, Tsk-1 mice crossed with 5-HT2B-/- mice showed reduced hypodermal thickening, reduced collagen content, and decreased numbers of myofibroblasts compared with TSk-1 mice crossed with 5-HT2B +/+ mice.
To confirm the link between platelet activation and increased 5-HT/5-HT2B signalling, we examined whether inhibition of platelet activation reduces tissue levels of 5-HT and prevents experimental fibrosis. Indeed, treatment with the P2Y12 receptor inhibitor clopidogrel reduced the content of 5-HT in fibrotic skin of bleomycin-challenged mice by 58 ± 21%. In parallel, clopidogrel decreased dermal thickening by 61 ± 13% as compared with control mice. This was also seen in Tsk-1 mice experiments, in which treatment with clopidogrel reduced hypodermal thickening by 51 ± 16%.
Tryptophan hydroxylase (TPH) 1 is the key enzyme for the synthesis of 5-HT in platelets. Experiments with TPH 1 deficient mice further underlined the important role of platelet-derived 5-HT in experimental fibrosis. Blood levels of 5-HT in TPH1-/- mice were reduced to 5% compared with wild-type animals [23]. Dermal thickening in bleomycin-challenged TPH1-/- mice were decreased by 61 ± 6% as compared with bleomycin-challenged TPH1+/+ mice. Similarly, collagen content and myofibroblast counts were significantly reduced.