TGF-B in Interstitial Lung Disease

描述（由申请人提供）：本提案旨在继续我们对选定肽生长因子介导间质性肺纤维化（IPF）的机制的研究。 我们发现，两种TNF-α受体均被敲除的小鼠表现出TGF-β 1表达降低，并且暴露于石棉、博来霉素和二氧化硅后未发生IPF。 我们实验室的新数据表明，通过用腺病毒载体（AdV）转导活性TGF-β 1的表达，可以在这些小鼠中重建疾病过程。此外，129近交系小鼠品系类似地表现出TGF-β 1表达降低，并且由于石棉暴露和用转导TGF-β 1的腺病毒载体治疗而显著减少的疾病。TGF-β 1是介导肺间质纤维化的关键分子。 虽然这一点在许多实验中已经明确，但TGF-β 1在IPF中的作用以及TGF-β 1介导其众所周知的作用的机制仍不明确。 因此，在我们工作的这一竞争性更新中，我们提出了以下假设：1）由TNF-α诱导并由肺泡上皮细胞和间充质细胞表达的TGF-β 1被活性氧从潜伏形式激活：2）生物活性TGF-β 1在体内限制上皮细胞增殖; 3）TGF-β 1诱导信号转导分子MAPK和IkappaB，导致TNF-α RKO小鼠中AP-1和NF-κ B活化，从而在这些纤维化抗性动物中诱导炎症和疾病; 4）TGF-β 1的反义cRNA将阻断疾病过程，为IPF提供“原理证明”和潜在的治疗方法。为了验证这一假设，我们提出了以下目的：目的1）为了证实我们的初步观察结果，即从AdV转导TGF-β 1表达的TNF-α RKO小鼠比正常背景小鼠表现出更严重和更长的间质纤维化，并且在这些小鼠和129品系中细支气管肺泡上皮的增殖受到TGF-β 1的抑制。 目的2）在细胞培养实验和用AdV-TGF-β 1构建体处理并暴露于石棉的小鼠体内，确定活性氧是否从潜伏形式激活TGF-β 1。 目的3）确定在来自TNF-α RKO小鼠的原代肺成纤维细胞中，TGF-β 1诱导的MAPK和IkappaB信号转导通路导致AP 1和NF-κ B活化，从而解释在这些纤维化抗性动物中观察到的炎症和疾病。 目的4）开发针对TGF-β 1和TNF-α的反义cRNA，其可在腺病毒载体系统中用作治疗剂，以在我们的动物模型中阻断IPF的发展。

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.