Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.

中文翻译：

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来自转染 bFGF 的骨骼肌细胞的组织蛋白酶 L 促进内皮细胞迁移。

已显示碱性成纤维细胞生长因子 (bFGF) 的基因转移可在缺血性疾病模型中诱导显着的内皮迁移和血管生成。在这里，我们调查转染 bFGF 基因的骨骼肌细胞 (SkMC) 分泌哪些因子以及它们是否参与内皮细胞迁移。我们构建了含有人 bFGF 基因 (Ad/bFGF) 或对照 LacZ 基因 (Ad/LacZ) 的复制缺陷型腺病毒载体，并从被 Ad/bFGF 或 Ad/ LacZ，分别。与用 LacZ-CM 孵育的细胞相比，用 bFGF-CM 孵育的 HUVEC 中的细胞迁移显着增加。有趣的是，响应 bFGF-CM 的 HUVEC 迁移仅被添加 bFGF 中和抗体部分阻断，表明 bFGF-CM 包含刺激内皮细胞迁移的其他因素。与 LacZ-CM 相比，bFGF-CM 中基质金属蛋白酶-1 (MMP-1)、纤溶酶原激活剂抑制剂-1 (PAI-1) 和组织蛋白酶 L 的几种蛋白质增加；基于一维凝胶电泳和质谱。RT-PCR 和免疫印迹分析证实了它们增加的 mRNA 和蛋白质水平。重组人 bFGF 蛋白诱导了 SkMCs 中 MMP-1、PAI-1 和组织蛋白酶 L 的表达。在用含有针对 MMP-1 或 PAI-1 的中和抗体的 bFGF-CM 处理的组中，内皮细胞迁移减少。特别是，用含有细胞不可渗透组织蛋白酶 L 抑制剂的 bFGF-CM 处理的 HUVEC 显示出细胞迁移最显着的减少。组织蛋白酶 L 蛋白通过 JNK 途径直接促进内皮细胞迁移。这些结果表明，转染 bFGF 基因的 SkMCs 释放的组织蛋白酶 L 可以促进内皮细胞迁移。