Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Polyclinic San Martino Hospital, Genoa, Italy.
Oncologic Surgery, Department of Surgery, Polyclinic San Martino Hospital, Genoa, Italy.
Arthritis Res Ther. 2018 May 2;20(1):77. doi: 10.1186/s13075-018-1577-0.
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.
Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3 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.
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.
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.
成纤维细胞通过过度产生细胞外基质（ECM）蛋白，主要是 I 型胶原（COL-1）和纤维连接蛋白（FN），导致纤维化。在系统性硬化症（SSc）中，这一过程由成纤维细胞内信号转导分子的激活介导，包括细胞外信号调节激酶 1 和 2（Erk1/2）和蛋白激酶 B（Akt）。塞来昔帕格是一种为治疗肺动脉高压而合成的前列环素受体激动剂。该研究探讨了塞来昔帕格及其活性代谢物（ACT-333679）下调 SSc 成纤维细胞/肌成纤维细胞原代培养中涉及的促纤维化活性和纤维化信号分子的可能性。
从获得伦理委员会（EC）批准并签署知情同意书的 SSc 患者皮肤活检中获取成纤维细胞，培养至第 3 代，然后在正常生长培养基中维持（未处理细胞）或分别用不同浓度的塞来昔帕格（30μM 至 0.3μM）或 ACT-333679（10μM 至 0.1μM）处理 48 小时。通过 Western blot 和定量实时 PCR 研究α-平滑肌肌动蛋白（α-SMA）、成纤维细胞特异性蛋白 1（S100A4）、COL-1 和 FN 的蛋白和基因表达。通过 Western blot 研究未处理和 ACT-333679 处理细胞中 Erk1/2 和 Akt 磷酸化。
与未处理细胞相比，塞来昔帕格和 ACT-333679 显著降低了培养的 SSc 成纤维细胞/肌成纤维细胞中α-SMA、S100A4 和 COL-1 的蛋白合成和基因表达，而 FN 的蛋白水平显著下调。有趣的是，ACT-333679 显著降低了培养的 SSc 成纤维细胞/肌成纤维细胞中 Erk1/2 和 Akt 的磷酸化。
塞来昔帕格及其主要活性代谢物 ACT-333679 首次被发现可能至少在体外通过下调成纤维细胞 Erk1/2 和 Akt 信号分子来干扰培养的 SSc 成纤维细胞/肌成纤维细胞的促纤维化活性。
