Treatment Strategy for Organ Fibrosis Targeting TGF-β and Smad Signaling

背景与目的：转化生长因子-β及其细胞内介质Smad蛋白在促进胶原基因转录方面发挥重要作用，可作为治疗器官纤维化的靶点。然而，对转化生长因子-β/Smad信号的干预也影响了生理信号转导，可能会对临床应用造成严重的不利影响。我们试图通过选择性地在胶原生成细胞中表达转化生长因子-β/SMAD拮抗剂来抑制肝纤维化。方法：将表达绿色荧光蛋白或转化生长因子-β/Smad信号抑制因子YB-1的重组腺病毒注射到未处理或经四氯化碳处理的小鼠体内。在激光共聚焦扫描显微镜下进行GFP荧光分析。通过荧光素酶活性测定和转基因报告鼠的组织学检查，检测YB-1过表达的抗纤维化作用。结果：以CAG表达单位为对照，GFP在正常肝和CCl_4处理的肝细胞中均有较强的表达。相反，小鼠α-2(I)胶原基因的组织特异性增强子(COL1A2)在CCl_4诱导的肝纤维化中激活的肝星状细胞中检测到绿色荧光蛋白的表达，而在未治疗的正常肝脏中则未检测到。当使用COL1A2增强子时，在其他任何器官中都没有观察到GFP荧光。COL1A2增强子控制下腺病毒介导的YB-1表达显著降低了CCl_4注射后COL1A2启动子的活性，从而抑制了肝纤维化的进展。结论：这些结果验证了一种治疗肝纤维化的新概念，即只在纤维化的肝脏实现细胞类型特异性基因的表达，而对其他器官的损害很小。

Background & Aims : TGF-β and its intracellular mediators, Smad proteins, play important roles in stimulating collagen gene transcription, and thus could be the targets for treating organ fibrosis. However, intervention of the TGF-β/Smad signal affects physiological signal transduction as well, and may cause serious adverse effects upon clinical application. We have attempted to suppress liver fibrosis by expressing a TGF-β/Smad antagonist selectively in collagen-producing cells only in the fibrotic liver. Methods : Recombinant adenoviruses expressing either GFP or a TGF-β/Smad signal repressor, YB-1, were injected to the mice untreated or treated with carbon tetrachloride (CCl_4). GFP fluorescence was analyzed under a confocal laser-scanning microscopy. Anti-fibrotic effects of YB-1 overexpression were examined by luciferase assays and histological examination using transgenic reporter mice. Results : When using the CAG expression unit as a control, GFP was strongly expressed in a large number of hepatocytes in both normal and CCl_4-treated liver. In contrast, GFP expression driven by a tissue-specific enhancer of the mouse α2(I) collagen gene (COL1A2) was detected in activated hepatic stellate cells in CCl_4-induced fibrotic liver, but not in untreated normal liver. There was no GFP fluorescence observed in any other organs when using the COL1A2 enhancer. Adenovirus-mediated YB-1 expression under the control of the COL1A2 enhancer significantly decreased COL1A2 promoter activity following CCl_4 injection and subsequently suppressed the progression of liver fibrosis. Conclusions : These results validate a new concept of the therapy for hepatic fibrosis to achieve cell type-specific gene expression only in the fibrotic liver with little damage to other organs.