RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells: implications for Duchenne muscular dystrophy.
RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells: implications for Duchenne muscular dystrophy.
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DOI：
10.1002/jcsm.12923
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Díaz-Manera J
中科院分区：
文献类型：
--
作者：
Fernández-Simón E;Suárez-Calvet X;Carrasco-Rozas A;Piñol-Jurado P;López-Fernández S;Pons G;Bech Serra JJ;de la Torre C;de Luna N;Gallardo E;Díaz-Manera J
Duchenne肌营养不良症(DMD)缺乏肌营养不良蛋白的表达，导致肌肉纤维，并被纤维脂肪组织替代。虽然一些生长因子在纤维化形成过程中的作用已经被研究，但PDGF-AA激活的通路到目前为止还没有描述。我们的目的是研究PDGF-AA在DMD纤维化过程中的分子作用。采用蛋白质组学方法对PDGF-AA处理后的3种DMD的骨骼肌成纤维脂肪前体细胞(FAPs)进行了分析。用免疫印迹、免疫荧光和G-LISA对质谱学结果进行确认。我们用C3-外酶和法舒地尔两种抑制剂评价了PDGF-AA对RhoA通路激活的影响。BrdU法和迁移实验检测细胞的增殖和迁移。肌动蛋白重组和胶原合成分别用鬼臼乙素染色和Sirol法进行检测。在一项体内概念验证研究中，我们用法舒地尔治疗dba/2J-MDX小鼠6周。用握力评定肌力。免疫荧光和流式细胞术分析研究肌肉组织中的纤维化和炎症标志物。质谱分析显示，治疗后DMD FAP患者RhoA途径蛋白表达上调(n=3，平均年龄=10.8±1.15岁)。蛋白质组学数据验证表明，与健康对照组相比，DMD肌肉中Arhgef2的表达显著增加，增加了7.7倍(n=2，平均年龄=10.8±1.14岁)。体外研究表明，RhoA/ROCK2通路可被血小板衍生生长因子-AA显著激活(n=，增加1.88倍，P<0.01)，而C3-胞外酶和法舒地尔均可阻断这种激活(n=0.05，P<0.001)。血小板衍生生长因子-AA激活RhoA通路可显著促进FAP的增殖和迁移(n=0.001，P<0.001)，而C3胞外酶和法舒地尔则在72h抑制FAP的增殖，在48h和72h抑制FAP的迁移(n=0.001)。体内研究表明，法舒地尔改善了肌肉功能(n=5非治疗dba/2j-mdx和n=6治疗dba/2j-mdx，增加了1.76倍，P<0.013)，组织学研究表明I型胶原表达面积减少了23%(n=5非治疗dba/2j-mdx和n=治疗dba/2j-mdx，P<0.01)。我们的结果提示PDGF-AA促进DMD患者FAP中RhoA通路的激活。这一途径可能参与FAPs的激活，促进其增殖、迁移和肌动蛋白重组，这代表着纤维化过程的开始。抑制RhoA通路可作为治疗肌营养不良患者肌肉纤维化的潜在靶点。
The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and replacement by fibro‐adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been studied, pathways activated by PDGF‐AA have not been described so far. Our aim was to study the molecular role of PDGF‐AA in the fibrotic process of DMD. Skeletal muscle fibro‐adipogenic progenitor cells (FAPs) from three DMD treated with PDGF‐AA at 50 ng/mL were analysed by quantitative mass spectrometry‐based proteomics. Western‐blot, immunofluorescence, and G‐LISA were used to confirm the mass spectrometry results. We evaluated the effects of PDGF‐AA on the activation of RhoA pathway using two inhibitors, C3‐exoenzyme and fasudil. Cell proliferation and migration were determined by BrdU and migration assay. Actin reorganization and collagen synthesis were measured by phalloidin staining and Sircol assay, respectively. In an in vivo proof of concept study, we treated dba/2J‐mdx mice with fasudil for 6 weeks. Muscle strength was assessed with the grip strength. Immunofluorescence and flow cytometry analyses were used to study fibrotic and inflammatory markers in muscle tissue. Mass spectrometry revealed that RhoA pathway proteins were up‐regulated in treated compared with non‐treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expression was significantly increased in DMD muscles compared with healthy controls by a 7.7‐fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF‐AA (n = 3, 1.88‐fold increase, P < 0.01) and both C3‐exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF‐AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3‐exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non‐treated dba/2J‐mdx and n = 6 treated dba/2J‐mdx, 1.76‐fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen‐I expression area (n = 5 non‐treated dba/2J‐mdx and n = 6 treated dba/2J‐mdx, P < 0.01). Our results suggest that PDGF‐AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies.
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