The results of the study show for the first time that selexipag and mainly its active metabolite ACT-333679 can potentially downregulate the profibrotic activity of cultured SSc fibroblasts/myofibroblasts through the reduction of Erk1/2 and Akt phosphorylation/activation at least in vitro.
The effect was characterized after treatment by the reduced synthesis of specific myofibroblast markers, such as α-SMA and S100A4, and then of ECM proteins, COL-1 and FN.
Of note, cultured SSc skin fibroblasts, maintained for 48 h in growth medium, expressed high levels of α-SMA compared to cultured HS skin fibroblasts, confirming their ongoing transition into activated myofibroblasts.
Therefore, the ability of selexipag and ACT-333679 to downregulate the gene expression and the protein synthesis of α-SMA and S100A4, seems to suggest a possible role for this IP receptor agonist to interfere with the fibroblast-to-myofibroblast transition.
It is recognized that in wound healing and in fibrotic diseases, including SSc, the transition of fibroblasts into profibrotic α-SMA+myofibroblasts is a fundamental event, making these cells the key mediators of fibrogenesis [28, 29]. The profibrotic capability of myofibroblasts is linked to their overproduction and deposition of ECM macromolecules, primarily COL-1 and FN, in the affected tissues [30, 31]. The present in vitro study confirmed that cultured SSc α-SMA+fibroblasts are characterized by greater ECM protein synthesis compared to cultured HS fibroblasts.
Therefore, the ability of selexipag and ACT-333679 to significantly reduce the protein synthesis of COL-1 and FN in cultured SSc skin fibroblasts/myofibroblasts might suggest a possible antifibrotic action of the IP receptor agonist.
Selexipag and ACT-333679 bind selectively and with higher affinity to the IP receptor compared to other prostacyclin analogs (such as beraprost or treprostinil), targeting the pathway of prostacyclin activation and leading to vasodilatory and anti-proliferative effects, as observed in several mouse models of PAH [20, 30, 31]. In addition, ACT-333679 is present at 3-fold to 4-fold higher levels and is approximately 37-fold more potent in inducing in vitro activation of human IP receptor than the parent compound, suggesting that the metabolite is the major one responsible for the selexipag effects in humans [32, 33]. In accordance with these observations, the results of the present in vitro study showed that in cultured SSc skin fibroblasts/myofibroblasts, ACT-333679 induced the same effects as selexipag, but at a concentration three times lower than that of the parent compound (selexipag 3 μM and 0.3 μM, ACT-333679 1 μM and 0.1 μM). The concentrations of selexipag and especially ACT-333679 were in accordance with several in vitro studies [15, 24,25,26]. Moreover, the lowest ACT-333679 concentration (0.1 μM) is in range with the values of its bioavailability which were well-tolerated by the HSs and were indicated by the area under the curve during a dose interval of administration (AUCt), as reported in several studies [25, 26].
It is interesting to note that selexipag and ACT-333679 did not have a dose-dependent effect in reducing the profibrotic activity of cultured SSc skin fibroblasts/myofibroblasts. A possible explanation of this might be related to the intrinsic variability in the cellular response to treatments with selexipag and ACT-333679 in cultured skin fibroblasts isolated from each enrolled patient with SSc. Therefore, the variability might be reduced by increasing the number of patients with SSc and the related experiments.
Another possible explanation for the absence of a dose-response effect might depend on the expression level and activity of the IP receptor on the cell surface of cultured SSc skin fibroblasts/myofibroblasts. In a recent study it has been observed that several cell types, including pulmonary arterial smooth muscle cells (PASMCs), displayed a moderate degree of constitutive IP receptor internalization and treatment with prostacyclin analogs (such as beraprost, treprostinil) induced strong depletion of this receptor from the cell surface, whereas the non-prostanoid agonists selexipag and ACT-333679 did not induce these effects [15]. Moreover, ACT-333679 1 μM and 10 μM induced the same increase in the expression of the IP receptor on the cell surface, which was higher than that induced by ACT-333679 0.1 μM [15]. Based on this observation, it might be possible to hypothesize that the concentrations of selexipag 0.3 μM and 3 μM and ACT-333679 0.1 μM and 1 μM might be sufficient to bind to all the IP receptors on the cell surface, inducing the activation of an intracellular signaling cascade that involves increase in cAMP production. On the other hand, ACT-333679 at the concentration of 0.1 μM, 1 μM, and 10 μM induced overlapping effects in terms of cAMP production and efficacy to contrast proliferation and fibrosis-related readouts in human PASMCs in a saturable manner, determining an efficacy plateau [15].
Another important result of our in vitro study is that selexipag and ACT-333679 exerted their effects efficiently targeting the profibrotic activity of fibroblasts/myofibroblasts of the skin, which represents one of the tissues affected by the fibrotic process. These observations suggest that prostacyclin pathways may play an important role in fibrotic diseases, including pulmonary fibrosis, as already observed for the prostacyclin analog iloprost, which reduces COL-1 synthesis through the increase in cAMP production in SSc skin fibroblasts [34].
FN is a multifunctional macromolecule produced by fibroblasts that can modulate fibroblast-mediated collagen gel contraction. According to this process, the activation of the IP receptor was recently shown to inhibit FN release and fibroblast-mediated collagen gel contraction, suggesting its possible contribution in tissue remodeling through the action on fibroblasts [35]. The effects of selexipag and ACT-333679, as observed at 48 h and limited to the reduction in protein synthesis, might suggest that their downregulatory effects on FN gene expression should be a very early event, as already observed in the in vitro study by Kamio et al. [35]. In this study the authors demonstrated how the prostacyclin analog beraprost induced downregulation of FN messenger RNA (mRNA) expression after 5 h of treatment, whereas the reduction in FN release was observed after 48 h of treatment [35]. This result might be due to the activation of post-transcriptional mechanisms induced by prostacyclin analogs and IP receptor agonists, which may be interesting to investigate. Based on this observation, even shorter in vitro experiments should be performed (at 4 h and 12 h).
Several molecules and growth factors contribute to the induction of the profibrotic phenotype of fibroblasts, including TGFβ1, ET-1 and cytokines (such as IL-6), through the activation of fibrogenic intracellular signaling pathways [7]. Among the molecules involved in these intracellular signaling pathways, Erk1/2 and Akt play an important role in mediating the effects of TGFβ1 and ET-1, which are considered key inducers of the fibrotic process and the levels of which are increased in SSc [7, 36]. As recently demonstrated, cultured SSc fibroblasts are characterized by increased phosphorylation of Erk1/2 and Akt when compared to HS fibroblasts suggesting the involvement of these molecules in mediating the activated profibrotic phenotype of SSc myofibroblasts [36].
The results of our in vitro study showed that cultured SSc fibroblasts/myofibroblasts are characterized by early phosphorylation and related activation of both Erk1/2 and Akt (15 min and 30 min) that was still present at a later time point (48 h).
Erk1/2 are members of the MAPK family implicated in several profibrotic signaling pathways [7, 37]. Previous investigations suggested that the activation of Erk1/2, induced by TGFβ1, is responsible for the overexpression of α-SMA and COL-1 in cultured fibroblasts, promoting the transition of these cells into profibrotic myofibroblasts [38]. In addition, several studies have reported that Erk1/2 can mediate the ET-1-induced expression of collagen isoforms and contractile proteins involved in the myofibroblast contraction and migration through the activation of the activator protein-1 (AP-1) transcription factor, contributing to the profibrotic effects of ET-1 [39,40,41].
Akt is a serine-threonine kinase that can engage multiple downstream signaling substrates and pathways, including the PI3K pathway, a Smad-independent intracellular signaling pathway that mediates TGFβ-induced fibrosis [7]. Very recent studies showed that the phosphorylation and the related activation of Akt are primarily implicated in fibroblast/myofibroblast differentiation, migration and proliferation of pulmonary and cardiac fibroblasts stimulated with TGFβ and ET-1 [42, 43].
Moreover, in previous investigations, the inhibition of PI3K/Akt phosphorylation was found to interfere with the expression of α-SMA in SSc lung fibroblasts and the ability of these cells to contract the collagen gel matrix mediated by ET-1 [44, 45].
Accordingly, the ability of ACT-333679 to significantly reduce the phosphorylation of both Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts found in our study, seems to further suggest that the effects of this IP receptor agonist on these cells might involve downregulation in the profibrotic signaling pathways.
One limitation of the study is that the antifibrotic effects of selexipag and ACT-333679 were investigated in in vitro cultures of human skin fibroblasts, so they should also be replicated on lung fibroblasts, since this IP receptor agonist was synthesized mainly for the treatment of PAH.
If confirmed on lung fibroblasts, the vasodilator activity exerted by selexipag in SSc patients with PAH should be integrated by important antifibrotic lung effects, possibly exerting disease-modifying effects over the long term. Moreover, to give an overview of selexipag and its vasodilator effect, it is necessary to cite a recent randomized, placebo-controlled phase II study that showed how selexipag did not reduce the number of Raynaud’s phenomenon (RP) attacks compared to placebo in adults with RP secondary to SSc, probably due to the differences between vascular effects in the systemic circulation compared to the pulmonary vasculature [46]. It is interesting to note that other therapies targeting the prostacyclin pathways can have positive or negative effects on RP based on the route of administration; in accordance with this observation, different studies highlighted that intravenous iloprost administration showed efficacy in reducing the number, severity, and duration of RP attacks whereas oral administration did not have any of these effects [47, 48]. Therefore, the route of administration of selexipag and other molecules targeting prostacyclin pathway may have an impact on the efficacy as indicated by the treatment response.
In addition, since Erk1/2 and Akt have been demonstrated to contribute to the intracellular signaling pathways responsible for the profibrotic effects exerted by TGFβ1 and ET-1, it might be interesting to investigate whether selexipag and ACT-333679 may contrast this action in cultured fibroblasts, both alone and in combination with selective TGFβ1 signaling blocking agents or ET-1 receptor antagonists, such as macitentan.
Moreover, based on present results, a new study is currently in progress to investigate the functional relevance of selexipag and its active metabolite, evaluating their ability to reduce the enhanced contractile fibrotic phenotype of SSc skin fibroblasts/myofibroblasts.