Effects of selexipag and its active metabolite in contrasting the profibrotic myofibroblast activity in cultured scleroderma skin fibroblasts

Abstract

Background: 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). 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.

Methods: Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3^rd 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.

Results: Selexipag 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.

Conclusions: Selexipag 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.

Keywords: Connective tissue diseases; Fibrosis; Prostacyclin receptor agonists; Skin fibroblasts; Systemic sclerosis.

Fig. 1

Evaluation of α-smooth muscle actin (α-SMA), type I collagen (COL-1), and fibronectin (FN) in cultured human skin fibroblasts isolated from patients with systemic sclerosis (SSc) and healthy subjects (HSs). a Immunocytochemistry analysis of α-SMA, COL-1 and FN in cultured human SSc and HS skin fibroblasts maintained in normal growth medium without treatment for 48 h. b Immunocytochemistry analysis of α-SMA in cultured human SSc skin fibroblasts maintained in normal growth medium (untreated), treated with selexipag at the concentration of 30 μM, 3 μM, and 0.3 μM, and treated with ACT-333679 at the concentration of 10 μM, 1 μM, and 0.1 μM for 48 h

Fig. 2

Evaluation of the protein synthesis of myofibroblast phenotype markers and extracellular matrix macromolecules in cultured human systemic sclerosis (SSc) skin fibroblasts. Western blotting and related densitometric analysis of the protein synthesis of α-smooth muscle actin (α-SMA), S100A4, type I collagen (COL-1) and fibronectin (FN) in cultured human SSc skin fibroblasts maintained in normal growth medium (untreated), treated with selexipag at the concentration of 30 μM, 3 μM, and 0.3 μM, and treated with ACT-333679 at the concentration of 10 μM, 1 μM, and 0.1 μM, for 48 h. For each experimental condition, the value of the synthesis of α-SMA, S100A4, COL-1, and FN was normalized to that of the corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The resulting value with each treatment was compared to that of the untreated cells (taken as a unit value), in order to obtain the level of protein synthesis. A molecular weight (MW) lane was also included. The final results represent the mean ± standard deviation (SD) of the values obtained from six independent experiments on cultured human SSc skin fibroblasts: *p < 0.05 and **p < 0.01 vs. untreated cells. kDa, kiloDalton

Fig. 3

Evaluation of the gene expression of myofibroblast phenotype markers and extracellular matrix macromolecules in cultured human systemic sclerosis (SSc) skin fibroblasts. Quantitative real-time polymerase chain reaction (qRT-PCR) and related analysis of the gene expression of α-smooth muscle actin (α-SMA), S100A4, type I collagen (COL-1) and fibronectin (FN) in cultured human SSc skin fibroblasts maintained in normal growth medium (untreated), treated with selexipag at the concentration of 30 μM, 3 μM, and 0.3 μM, and treated with ACT-333679 at the concentration of 10 μM, 1 μM, and 0.1 μM, for 48 h. The final results represent the mean ± standard deviation (SD) of the values obtained from six independent experiments on cultured human SSc skin fibroblasts: *p < 0.05 and **p < 0.01 vs. untreated cells

Fig. 4

Evaluation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt) activation in cultured human systemic sclerosis (SSc) skin fibroblasts. a Western blotting and related densitometric analysis of phospho-Erk1/2 (p-Erk1/2), Erk1/2, phospho-Akt (p-Akt) and Akt in cultured human SSc skin fibroblasts maintained in normal growth medium (untreated) or treated with ACT-333679 at the concentration of 10 μM, 1 μM, and 0.1 μM for 48 h. The expression of phosphorylated proteins (p-Erk1/2 and p-Akt) was first normalized to that of the naïve proteins (Erk1/2 and Akt) and then normalized to that of the related expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The resulting value of each treatment was compared to that of the untreated cells (taken as the unit value): *p < 0.05 vs. untreated cells. b Western blotting and related densitometric analysis of phospho-Erk1/2 (p-Erk1/2), Erk1/2, phospho-Akt (p-Akt) and Akt, in cultured human SSc skin fibroblasts maintained in normal growth medium (untreated) or treated with ACT-333679 at the concentration of 10 μM, 1 μM, and 0.1 μM for 15 min and 30 min. The expression of phosphorylated proteins (p-Erk1/2 and p-Akt) was normalized to that of the naïve proteins (Erk1/2 and Akt). The resulting value was then normalized to that of the related expression of GAPDH: ^#p < 0.01 vs. time zero (T0); *p < 0.05 and **p < 0.01 vs. untreated cells. The final results represent the mean ± standard deviation (SD) of the values obtained from six independent experiments on cultured human SSc skin fibroblasts