The Role of TGF-Beta in Acute Lung Injury

描述（由申请人提供）：我们希望确定导致急性肺损伤（ALI）发生的分子机制。这种毁灭性的临床综合征的特征是形成肺水肿-肺泡充满富含蛋白质的渗出物。最近，我们的实验室已经确定了细胞因子转化生长因子-β（TGF-β）作为ALI中可能的关键介质;抑制TGF-β可以防止小鼠模型中肺水肿的发展。其他研究人员已经表明，TGF-β在体外直接增加内皮通透性。我们已经发现，TGF-β通过与细胞内谷胱甘肽（GSH）耗竭相关的机制直接增加上皮通透性。我们假设局部活化的TGF-β通过对上皮细胞和内皮细胞产生特异性作用，介导了ALI相关肺水肿的发生。此外，我们假设TGF-β通过改变细胞内巯基氧化还原状态的机制改变内皮和上皮细胞连接蛋白的磷酸化状态。连接蛋白磷酸化会影响连接的完整性，因此，细胞旁通透性的程度。我们将使用TGF-β绿色荧光蛋白报告小鼠，以确定个别居民上皮和内皮细胞，刺激TGF-β在体内。将使用上皮和内皮细胞单层评估TGF-β对细胞旁通透性的影响。将测量还原型和氧化型GSH，以确定巯基氧化还原状态。将确定上皮和内皮连接蛋白磷酸化状态并鉴定特异性蛋白。与大多数其他可能的ALT治疗方法（仅在损伤发生前有效）相反，TGF-β抑制剂即使在损伤发生后也能有效地减弱ALT（先前在我们的实验室中显示）。因此，鉴定TGF-β影响上皮和/或内皮细胞的特定分子机制可能导致治疗ALI（一种常见且通常致命的临床综合征）的新的和改进的策略。拟议的研究还将帮助我发展和获得必要的技能，以实现我成为一名成功和独立的肺部疾病科学家的最终目标。

DESCRIPTION (provided by applicant): We wish to identify molecular mechanisms leading to the development of acute lung injury (ALI). This devastating clinical syndrome is characterized by formation of pulmonary edema--alveolar flooding with a protein-rich exudate. Recently, our laboratory has identified the cytokine transforming growth factor-Beta (TGF-Beta) as a possible critical mediator in ALI; inhibition of TGF-Beta prevents the development of pulmonary edema in murine models. Other investigators have shown that TGF-Beta directly increases endothelial permeability in vitro. We have found that TGF-Beta directly increases epithelial permeability by a mechanism that is associated with depletion of intracellular glutathione (GSH). We hypothesize that locally activated TGF-Beta mediates the development of pulmonary edema associated with ALI by exerting specific effects on resident epithelial and endothelial cells. Furthermore, we hypothesize that TGF-Beta alters the phosphorylation state of endothelial and epithelial cell junction proteins by mechanisms involving changes in intracellular thiol redox status. Junction protein phosphorylation would affect junction integrity, and therefore, the degree of paracellular permeability. We will use TGF-Beta green fluorescent protein reporter mice to identify individual resident epithelial and endothelial cells that are stimulated by TGF-Beta in vivo. Epithelial and endothelial cell monolayers will be used to assess TGF-Beta effect on paracellular permeability. Reduced and oxidized GSH will be measured to determine thiol redox status. Epithelial and endothelial junction protein phosphorylation state will be determined and specific proteins identified. In contrast to most other potential treatments for ALT, which are effective only if administered before the onset of injury, TGF-Beta inhibition effectively attenuates ALT even after the onset of injury (previously shown in our laboratory). Therefore, identification of specific molecular mechanisms by which TGF-Beta affects epithelial and/or endothelial cells could lead to novel and improved strategies to treat ALI, a common and often lethal clinical syndrome. The proposed studies will also help me develop and acquire the necessary skills to achieve my ultimate goal of becoming a successful and independent scientist in pulmonary disease.