TGF-B in Interstitial Lung Disease

描述(由申请人提供)：这项建议旨在继续我们关于选定的肽生长因子介导间质性肺纤维化(IPF)的机制的工作。我们发现，两种受体都被敲除的小鼠表现出转化生长因子-β1的表达减少，并且由于暴露于石棉、博莱霉素和二氧化硅而未能发生肺间质纤维化。我们实验室的新数据表明，用转导活性转化生长因子-β1表达的腺病毒载体(ADV)治疗这些动物，可以在这些小鼠中重建疾病过程。此外，129近交系小鼠在石棉暴露和转导转化生长因子-β1的腺载体治疗后，同样表现出转化生长因子-β1的表达降低和疾病显著减少。转化生长因子-β1是介导间质性肺纤维化的关键分子。虽然这一点已经在许多实验中得到了明确，但转化生长因子-β1在IPF中扮演的角色以及转化生长因子-β1介导其众所周知的影响的机制仍不清楚。因此，在我们工作的竞争性更新中，我们提出了假设：1)由TNF-α诱导的肺泡上皮细胞和间充质细胞表达的转化生长因子-β1是被活性氧从潜伏期激活的；2)生物活性的转化生长因子-β1在体内限制了上皮细胞的增殖；3)转化生长因子-β1诱导了信号转导分子MAPK和IkappaB的激活，导致了AP-1和NF-kappaB的激活，从而导致了这些纤维化抵抗动物的炎症和疾病；4)针对转化生长因子-β1的反义cRNA阻断了IPF的发病过程，为IPF的治疗提供了“理论依据”和潜在的治疗途径。为了验证这一假说，我们提出了以下目标：1)为了证实我们的初步观察结果，转导转化生长因子-β1的肿瘤坏死因子-αRKO小鼠表现出比正常背景小鼠更严重和更持久的间质纤维化，并且转化生长因子-β1抑制这些小鼠和129株小鼠细支气管肺泡上皮细胞的增殖。目的2)在细胞培养实验和体内实验中，确定细胞培养实验和体内实验中，活性氧是否能从潜伏期激活转化生长因子-β1(TGF-β1)。目的3)研究转化生长因子-β1诱导的小鼠肺成纤维细胞中MAPK和IkappaB活化的信号转导途径，从而解释这些纤维化抵抗动物的炎症和疾病。目的4)研制针对转化生长因子-β1和肿瘤坏死因子-α的反义CRNAs，用于腺载体系统，阻断动物模型肺间质纤维化的发生发展。

DESCRIPTION (provided by applicant): This proposal is designed to continue our work on the mechanisms through which selected peptide growth factors mediate interstitial pulmonary fibrosis (IPF). We showed that mice with both receptors for TNF-alpha knocked out exhibited reduced TGF-beta1 expression and failed to develop IPF consequent to exposure to asbestos, bleomycin, and silica. New data from our laboratory show that the disease process can be reestablished in these mice by treating the animals with an adenovirus vector (AdV) transducing the expression of active TGF-beta1. In addition, the 129 inbred mouse strain similarly exhibits reduced TGF-beta1 expression and significantly reduced disease consequent to asbestos exposure and treatment with the adenovector transducing TGF-beta1. TGF-beta1 is a key molecule mediating interstitial pulmonary fibrosis. While this has been made clear in many experiments, the role that TGF-beta1 plays in IPF and the mechanisms through which TGF-beta1 mediates its well-known effects remain undefined. Thus, in this competitive renewal of our work, we put forth the Hypothesis: that 1) TGF- beta1 induced by TNF-alpha and expressed by alveolar epithelial and mesenchymal cells is activated from the latent form by reactive oxygen species; that 2) the biologically active TGF-beta1 limits epithelial proliferation in vivo; that 3) TGF-beta1 induces the signal transduction molecules MAPK and IkappaB leading to AP-1 and NF-kappaB activation in the TNF-alphaRKO mice thus inducing inflammation and disease in these fibrogenic-resistant animals; and that 4) an antisense cRNA to TGF-beta1 will block the disease process, provide a "proof of principle" and a potential therapeutic approach for IPF. To test this hypothesis, we propose the following Aims: Aim 1) To confirm our preliminary observations showing that TNF-alphaRKO mice transducing TGF-beta1 expression from the AdV exhibit more severe and prolonged interstitial fibrogenesis than normal background mice, and that proliferation of the bronchiolar-alveolar epithelium is inhibited by TGF-beta1 in these mice and in the 129 strain. Aim 2) To determine whether or not reactive oxygen species activate TGF-beta1 from the latent form in cell culture experiments and in vivo in mice treated with the AdV- TGF-beta1 construct and exposed to asbestos. Aim 3) To determine, in primary lung fibroblasts from TNF-alphaRKO mice, TGF-beta1 -induced MAPK and IkappaB signal transduction pathways leading to AP1 and NF-kappaB activation, thus explaining the inflammation and disease observed in these fibrogenic-resistant animals. Aim 4) To develop anti-sense cRNAs, against TGF-beta1 and TNF-alpha that can be used in an adenovector system as therapeutic agents to block the development of IPF in our animal models.