Fibroblast control of TGF-beta activation by Thy-1 and lung fibrosis

描述(申请人提供)：特发性肺纤维化是一种罕见的、进行性的、致命的疾病，目前还没有有效的治疗方法。在特发性肺纤维化中，成纤维细胞亚群对伤口愈合的调节失调会导致纤维化。转化生长因子-β是伤口愈合反应的关键调节因子，其作用在纤维化进展中起关键作用。转化生长因子-β必须从潜伏期被激活才能影响这些反应。我们的数据表明，成纤维细胞亚群的不同特征之一是能够激活潜在的转化生长因子-1来应对损伤。肺成纤维细胞上GPI锚定蛋白Thy-1的存在限制了其激活转化生长因子-(。这些数据表明，Thy-1是一种生物反应调节剂，可以限制转化生长因子-1的激活。博莱霉素刺激的THY-1(-)成纤维细胞表达潜伏的转化生长因子结合蛋白4(LTBP-4)的水平高于THY-1()细胞，这与LTBP-4亚型小鼠肺组织中的转化生长因子-4活性增加一致。THY-1(-)LTBP-4亚型肺成纤维细胞对博莱霉素的反应不能激活潜伏的转化生长因子-α，提示LTBP-4对Thy-1(-)细胞的应答能力是必需的。由于转化生长因子-α的活性在肺纤维化的形成过程中起关键作用，间质成纤维细胞激活潜伏的转化生长因子-β的能力可能是肺损伤良性消退或纤维化并发症发生的关键决定因素。我们假设肺成纤维细胞激活潜在的转化生长因子的能力对肺损伤后纤维化的进展至关重要。因此，具有减弱的转化生长因子-1激活的成纤维细胞将有利于有限的伤口愈合反应，而在损伤反应中表现出强大的转化生长因子-1激活的成纤维细胞将有利于纤维化反应。这项建议的总体目标是确定肺成纤维细胞激活的转化生长因子-1是如何影响肺纤维化的进展的。我们将使用一种新的策略，利用改良腺病毒和成纤维细胞特异性启动子在体内驱动成纤维细胞特异性转基因Thy-1和LTBP-4的表达，以测试成纤维细胞特异性表达这些蛋白在博莱霉素诱导的小鼠肺纤维化模型中对纤维化进展的影响。我们提出以下目的：特定目的1：验证成纤维细胞特异性过表达Thy-1通过限制转化生长因子-1的激活来保护博莱霉素诱导的肺纤维化的假说，并确定固有活性的转化生长因子-1的表达是否取消了过度表达Thy-1的潜在保护作用；特定目的2：验证成纤维细胞表达LTBP-4对于潜伏性转化生长因子-1的激活是必需的假说，以及LTBP-4是否在体内取消了对Thy-1的保护。这些研究将为肺成纤维细胞亚群如何决定纤维化进展提供新的见解，并可能确定治疗特发性肺纤维化的新治疗方法。

DESCRIPTION (provided by applicant): Idiopathic pulmonary fibrosis is a rare and progressive, fatal disease for which there is no effective treatment. Dysregulated wound healing by subsets of fibrogenic lung fibroblasts leads to fibrosis in idiopathic pulmonary fibrosis. TGF-( is a key regulator of wound healing responses and its action is critical for fibrogenic progression. TGF-( must be activated from its latent state to affect these responses. Our data show that one of the differential characteristics of fibroblast subsets is the capacity to activate latent TGF-( in response to injury. The presence of the GPI-anchored protein, Thy-1, on lung fibroblasts limits the ability to activate TGF-(. These data suggest that Thy-1 is a biological response modifier which limits TGF-( activation. Thy-1 (-) fibroblasts express higher levels of the latent TGF-( binding protein 4 (LTBP-4) when stimulated with bleomycin than do Thy-1 (+) cells, consistent with the increased TGF-( activity in the lungs of LTBP-4 hypomorphic mice. Thy-1 (-) LTBP-4 hypomorphic lung fibroblasts fail to activate latent TGF-( in response to bleomycin, suggesting that LTBP-4 is necessary for the ability of Thy-1 (-) cells to respond. Since TGF-( activity is critical to the fibrogenic progression of pulmonary fibrosis, the capacity of interstitial fibroblasts to activate latent TGF-( could be a key determinant of either benign resolution of lung injury or the development of fibrotic complications. We hypothesize that the ability of pulmonary fibroblasts to activate latent TGF-( in response to stimulation is critical to fibrogenic progression following lung injury. Thus fibroblasts with attenuated TGF-( activation would favor limited wound healing responses, whereas, fibroblasts that exhibit robust TGF-( activation in response to injury would favor fibrogenic responses. The overall goal of this proposal is to determine how modulating TGF-( activation by lung fibroblasts affects progression of pulmonary fibrosis. We will use a novel strategy that employs a modified adenovirus and a fibroblast-specific promoter to drive fibroblast specific transgene expression of Thy-1 and LTBP-4 in vivo to test the effects of fibroblast-specific expression of these proteins on fibrogenic progression in a mouse model of bleomycin-induced pulmonary fibrosis. We propose the following aims: Specific Aim 1: To test the hypothesis that fibroblast-specific over-expression of Thy-1 protects from bleomycin-induced pulmonary fibrosis by limiting TGF-( activation and to determine if expression of constitutively active TGF-( abrogates the potential protective effects of Thy-1 overexpression; Specific Aim 2: To test the hypothesis that fibroblast expression of LTBP-4 is necessary for activation of latent TGF-( in vitro and whether LTBP-4 abrogates Thy-1 protection in vivo. These studies will provide new insights into how lung fibroblast subpopulations determine fibrogenic progression and potentially identify new therapeutic approaches to treat idiopathic pulmonary fibrosis.