Abstract
BackgroundMyofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved.MethodsFibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 μM to 0.3 μM) or ACT-333679 (from 10 μM to 0.1 μM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting.ResultsSelexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts.ConclusionsSelexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts/myofibroblasts at least in vitro, possibly through the downregulation of fibrogenic Erk1/2 and Akt signaling molecules.
Highlights
Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt)
Myofibroblast transition and ECM overproduction are known to be induced by several profibrotic mediators, including transforming growth factor-β (TGFβ), endothelin1 (ET-1), cytokines and chemokines, whose circulating levels are increased in patients with SSc
This study investigated the effects of selexipag and ACT-333679 in reducing the activity of cultured skin SSc fibroblasts and their ECM protein overproduction, through the ability to interfere with the activation of those intracellular signaling transduction molecules involved in the regulation and progression of fibrosis, primarily mitogen-activated protein kinase (MAPK) family members (i.e. extracellular signal-regulated kinases 1 and 2 (Erk1/2)) and protein kinase B (PKB or Protein kinase B (Akt))
Summary
Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). These mediators have been shown to regulate the progression of fibrosis through the activation of intracellular signaling transduction pathways, such as those involving the activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt and Wnt/β-catenin signaling [7,8,9,10] Based on this knowledge, the blockage of fibroblast-to-myofibroblast transition and the attenuation of the profibrotic myofibroblast activity and related ECM overproduction might represent important steps in reducing the fibrotic process at least in SSc [11, 12]
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