Objectives In systemic sclerosis (SSc), angiogenesis impairment advances in parallel with the development of fibrosis orchestrated by myofibroblasts originating from different sources, including endothelial-to-mesenchymal transition (EndoMT). Soluble guanylate cyclase (sGC) stimulation has shown antifibrotic effects in SSc skin fibroblasts and mouse models. Here, we investigated the effects of pharmacological sGC stimulation on impaired angiogenesis and myofibroblast-like features of SSc dermal microvascular endothelial cells (SSc-MVECs). Methods To determine whether sGC stimulation affected cell viability/proliferation, SSc-MVECs and healthy dermal MVECs (H-MVECs) were challenged with the sGC stimulator (sGCS) MK-2947 and assayed by annexin V/PI flow cytometry and WST-1. To study angiogenesis and EndoMT, MK-2947-treated SSc-MVECs were subjected to wound healing and capillary morphogenesis assays and analysed for the expression of endothelial/myofibroblast markers and contractile ability. Results MK-2947 treatment did not affect H-MVEC viability/proliferation, while it significantly increased SSc-MVEC proliferation, wound healing capability and angiogenic performance. After MK-2947 treatment, SSc-MVECs exhibited significantly increased proangiogenic MMP9 and decreased antiangiogenic MMP12 and PTX3 gene expression. A significant increase in the expression of CD31 and vascular endothelial cadherin paralleled by a decrease in α-smooth muscle actin, S100A4, type I collagen and Snail1 mesenchymal markers was also found in MK-2947-treated SSc-MVECs. Furthermore, stimulation of sGC with MK-2947 significantly counteracted the intrinsic ability of SSc-MVECs to contract collagen gels and reduced phosphorylated-ERK1/2 protein levels. Conclusion These findings demonstrate for the first time that pharmacological sGC stimulation effectively ameliorates the angiogenic performance and blunts the myofibroblast-like profibrotic phenotype of SSc-MVECs, thus providing new evidence for repurposing sGCSs for SSc.

中文翻译：

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可溶性鸟苷酸环化酶刺激促进血管生成并减弱系统性硬化症内皮细胞的肌成纤维细胞样特征

目的 在系统性硬化症 (SSc) 中，血管生成损伤与纤维化的发展并行，纤维化由不同来源的肌成纤维细胞协调，包括内皮-间质转化 (EndoMT)。可溶性鸟苷酸环化酶 (sGC) 刺激已在 SSc 皮肤成纤维细胞和小鼠模型中显示出抗纤维化作用。在这里，我们研究了药理学 sGC 刺激对 SSc 真皮微血管内皮细胞 (SSc-MVEC) 受损血管生成和肌成纤维细胞样特征的影响。方法 为了确定 sGC 刺激是否影响细胞活力/增殖，SSc-MVEC 和健康真皮 MVEC (H-MVEC) 受到 sGC 刺激物 (sGCS) MK-2947 的攻击，并通过膜联蛋白 V/PI 流式细胞术和 WST-1 进行测定。研究血管生成和 EndoMT，对 MK-2947 处理的 SSc-MVEC 进行伤口愈合和毛细血管形态发生测定，并分析内皮/肌成纤维细胞标记物的表达和收缩能力。结果 MK-2947 处理不影响 H-MVEC 的活力/增殖，而它显着增加 SSc-MVEC 增殖、伤口愈合能力和血管生成性能。MK-2947 处理后，SSc-MVEC 表现出显着增加的促血管生成 MMP9 和降低的抗血管生成 MMP12 和 PTX3 基因表达。在 MK-2947 处理的 SSc-MVEC 中还发现 CD31 和血管内皮钙粘蛋白的表达显着增加，同时 α-平滑肌肌动蛋白、S100A4、I 型胶原蛋白和 Snail1 间充质标记物的减少。此外，用 MK-2947 刺激 sGC 显着抵消了 SSc-MVEC 收缩胶原凝胶和降低磷酸化 ERK1/2 蛋白水平的内在能力。结论 这些发现首次表明，药理学 sGC 刺激可有效改善血管生成性能并减弱 SSc-MVEC 的肌成纤维细胞样促纤维化表型，从而为将 sGCS 重新用于 SSc 提供了新证据。