Neuromuscular Diseases and Neuroimmunology Unit, Foundation Neurological Institute "C. Besta", Milan, Italy.
Cell Tissue Res. 2010 Feb;339(2):397-410. doi: 10.1007/s00441-009-0889-4. Epub 2009 Nov 10.
To probe pro-fibrotic mechanisms in dystrophic muscle, we isolated primary fibroblasts from Duchenne muscular dystrophy (DMD) and control muscle biopsies and induced transdifferentiation in myofibroblasts by transforming growth factor beta1 (TGF-beta1) treatment. We compared proliferating activity, soluble collagen production, and transcript and protein levels of decorin, myostatin, TGF-beta1, matrix metalloproteinase-1 (MMP-1; interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin), MMP-7 (matrilysin), and the tissue inhibitors of metalloproteinases inhibitors (TIMPs) 1-4, in fibroblasts and myofibroblasts. Principal differences included a significantly greater proliferation rate and soluble collagen production, a significant upregulation of decorin, myostatin and MMP-7 transcripts and proteins, and a significant downregulation of MMP-1 and TIMP-3 transcripts (with MMP-1 protein being reduced as shown by enzyme-linked immunosorbent assay and TIMP-3 protein apparently being reduced on Western blot), in untreated DMD fibroblasts compared with controls. TGF-beta1 transdifferentiation significantly lowered decorin and myostatin and significantly increased TGF-beta1 transcript and protein, significantly increased MMP-1 and TIMP-3, and significantly lowered MMP-7 transcript and protein in DMD cells compared with pretreatment controls. The differences between DMD and control fibroblasts showed that DMD fibroblasts had a profibrotic phenotype, accentuated by TGF-beta1 treatment. Dystrophin absence itself could exert a direct influence on the homeostasis of the extracellular matrix (ECM) by allowing leakage of cellular components to the extracellular space or by abnormal cellular uptake of extracellular growth factors, cytokines, or enzymes influencing muscle fibroblasts either directly by altering adhesion properties or indirectly by interactions with molecules released into the ECM by muscle or inflammatory cells. The transdifferentiation of muscle fibroblasts might serve as a simplified model of fibrosis for further elucidation of the mechanisms of muscle fibrosis and for testing possible anti-fibrotic agents.
为了探究营养不良性肌肉中的促纤维化机制，我们从杜氏肌营养不良症（DMD）和对照肌肉活检中分离出原代成纤维细胞，并通过转化生长因子β1（TGF-β1）处理诱导成肌纤维细胞的转分化。我们比较了增殖活性、可溶性胶原产生以及核心蛋白聚糖、肌肉生长抑制素、TGF-β1、基质金属蛋白酶-1（MMP-1；间质胶原酶）、MMP-2（明胶酶）、MMP-3（基质溶解素）、MMP-7（基质金属蛋白酶-7；基质溶解素）和金属蛋白酶组织抑制剂（TIMP）1-4 的转录物和蛋白水平，在成纤维细胞和成肌纤维细胞中。主要差异包括增殖率和可溶性胶原产生显著增加，核心蛋白聚糖、肌肉生长抑制素和 MMP-7 转录物和蛋白显著上调，MMP-1 和 TIMP-3 转录物显著下调（酶联免疫吸附试验显示 MMP-1 蛋白减少，Western blot 显示 TIMP-3 蛋白明显减少），与对照组相比，未处理的 DMD 成纤维细胞。TGF-β1 转分化显著降低了核心蛋白聚糖和肌肉生长抑制素，显著增加了 TGF-β1 转录物和蛋白，显著增加了 MMP-1 和 TIMP-3，并且显著降低了 DMD 细胞中 MMP-7 转录物和蛋白与预处理对照相比。DMD 成纤维细胞与对照成纤维细胞之间的差异表明，DMD 成纤维细胞具有促纤维化表型，TGF-β1 处理进一步加重了这种表型。肌营养不良蛋白的缺失本身可能通过允许细胞成分漏出细胞外空间或通过异常摄取影响肌肉成纤维细胞的细胞外生长因子、细胞因子或酶，对细胞外基质（ECM）的内稳态产生直接影响，这些因子或酶直接通过改变粘附特性或通过与肌肉或炎症细胞释放到 ECM 中的分子相互作用来影响肌肉成纤维细胞。肌肉成纤维细胞的转分化可能作为纤维化的简化模型，用于进一步阐明肌肉纤维化的机制，并测试可能的抗纤维化药物。
